Naive Reversions No BP

In [1]:

import os
import tempfile
cache_dir = tempfile.mkdtemp()
os.environ['XDG_CACHE_HOME'] = cache_dir
import re
import numpy as np
import pandas as pd
import matplotlib.pyplot as plt
import seaborn as sns
import itertools
import warnings
import Bio.PDB
import piecewise_regression
import statsmodels.api as sm
from scipy.stats import spearmanr

import utils.trees as ut
import utils.phenotype_colorscales as pc
from utils.plotting import regplot_lowess_ci

import os import tempfile cache_dir = tempfile.mkdtemp() os.environ['XDG_CACHE_HOME'] = cache_dir import re import numpy as np import pandas as pd import matplotlib.pyplot as plt import seaborn as sns import itertools import warnings import Bio.PDB import piecewise_regression import statsmodels.api as sm from scipy.stats import spearmanr import utils.trees as ut import utils.phenotype_colorscales as pc from utils.plotting import regplot_lowess_ci

In [2]:

parameters

final_variant_scores = "data/dms/final_variant_scores.csv"
dms_sites = "data/dms/CGGnaive_sites.csv"
chigy_hc_mut_rates = "../passenger/output/chigy_hc_mutation_rates_nt.csv"
chigy_lc_mut_rates = "../passenger/output/chigy_lc_mutation_rates_nt.csv"
pdb = "data/AbCGG_structure/combined_ch2_eh2-coot_IMGT.pdb"
results = "../nextflow/results/"
ranking_subdir = 'naive_reversions_first'
metadata_csv = "../gc_metadata.csv"
outbase = "output/mutations"
workflow_env_exec = False # only set to True within the Nextflow papermill process

final_variant_scores = "data/dms/final_variant_scores.csv" dms_sites = "data/dms/CGGnaive_sites.csv" chigy_hc_mut_rates = "../passenger/output/chigy_hc_mutation_rates_nt.csv" chigy_lc_mut_rates = "../passenger/output/chigy_lc_mutation_rates_nt.csv" pdb = "data/AbCGG_structure/combined_ch2_eh2-coot_IMGT.pdb" results = "../nextflow/results/" ranking_subdir = 'naive_reversions_first' metadata_csv = "../gc_metadata.csv" outbase = "output/mutations" workflow_env_exec = False # only set to True within the Nextflow papermill process

In [3]:

injected-parameters

# Parameters
final_variant_scores = "final_variant_scores.csv"
dms_sites = "CGGnaive_sites.csv"
chigy_hc_mut_rates = "chigy_hc_mutation_rates_nt.csv"
chigy_lc_mut_rates = "chigy_lc_mutation_rates_nt.csv"
pdb = "combined_ch2_eh2-coot_IMGT.pdb"
results = "."
ranking_subdir = "naive_reversions_no_bp"
metadata_csv = "gc_metadata.csv"
outbase = "."
workflow_env_exec = True

# Parameters final_variant_scores = "final_variant_scores.csv" dms_sites = "CGGnaive_sites.csv" chigy_hc_mut_rates = "chigy_hc_mutation_rates_nt.csv" chigy_lc_mut_rates = "chigy_lc_mutation_rates_nt.csv" pdb = "combined_ch2_eh2-coot_IMGT.pdb" results = "." ranking_subdir = "naive_reversions_no_bp" metadata_csv = "gc_metadata.csv" outbase = "." workflow_env_exec = True

In [4]:

if not workflow_env_exec:
    plt.rcParams['font.sans-serif'] = 'Helvetica'

if not workflow_env_exec: plt.rcParams['font.sans-serif'] = 'Helvetica'

In [5]:

output_dir = f"{outbase}/{ranking_subdir}"
if not os.path.exists(output_dir):
    os.makedirs(output_dir)

output_dir = f"{outbase}/{ranking_subdir}" if not os.path.exists(output_dir): os.makedirs(output_dir)

DMS single AA mutant effects on affinity¶

In [6]:

# dms_df = pd.read_csv("https://media.githubusercontent.com/media/jbloomlab/Ab-CGGnaive_DMS/main/results/final_variant_scores/final_variant_scores.csv", index_col="mutation", dtype=dict(position_IMGT=pd.Int16Dtype()))
dms_df = pd.read_csv(final_variant_scores, index_col="mutation", dtype=dict(position_IMGT=pd.Int16Dtype()))
# remove linker sites
dms_df = dms_df[dms_df.chain != "link"]
# add indicator for wildtype data
dms_df["WT"] = dms_df.wildtype == dms_df.mutant
assert dms_df.position_IMGT.max() < 1000
dms_df["site"] = [f"{chain}-{str(pos).zfill(3)}" for chain, pos in zip(dms_df.chain, dms_df.position_IMGT)]
dms_df

# dms_df = pd.read_csv("https://media.githubusercontent.com/media/jbloomlab/Ab-CGGnaive_DMS/main/results/final_variant_scores/final_variant_scores.csv", index_col="mutation", dtype=dict(position_IMGT=pd.Int16Dtype())) dms_df = pd.read_csv(final_variant_scores, index_col="mutation", dtype=dict(position_IMGT=pd.Int16Dtype())) # remove linker sites dms_df = dms_df[dms_df.chain != "link"] # add indicator for wildtype data dms_df["WT"] = dms_df.wildtype == dms_df.mutant assert dms_df.position_IMGT.max() < 1000 dms_df["site"] = [f"{chain}-{str(pos).zfill(3)}" for chain, pos in zip(dms_df.chain, dms_df.position_IMGT)] dms_df

Out[6]:

	target	wildtype	position	position_IMGT	chain	annotation	mutant	codon	single_nt	bind_CGG	delta_bind_CGG	n_bc_bind_CGG	n_libs_bind_CGG	expr	delta_expr	n_bc_expr	n_libs_expr	WT	site
mutation
E1(H)A	CGG_naive	E	1	1	H	FWRH1	A	GAG	True	8.74239	0.11933	20	2	10.45318	-0.00246	20	2	False	H-001
E1(H)C	CGG_naive	E	1	1	H	FWRH1	C	GAG	False	8.60813	-0.01492	22	2	10.33348	-0.12216	22	2	False	H-001
E1(H)D	CGG_naive	E	1	1	H	FWRH1	D	GAG	True	8.63554	0.01249	18	2	10.50438	0.04874	18	2	False	H-001
E1(H)E	CGG_naive	E	1	1	H	FWRH1	E	GAG	True	8.62305	0.00000	23285	2	10.45565	0.00000	23285	2	True	H-001
E1(H)F	CGG_naive	E	1	1	H	FWRH1	F	GAG	False	8.75738	0.13433	29	2	10.34185	-0.11379	29	2	False	H-001
...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...
R128(L)S	CGG_naive	R	235	128	L	FWRL4	S	CGT	True	8.63400	0.01095	45	2	10.52908	0.07344	45	2	False	L-128
R128(L)T	CGG_naive	R	235	128	L	FWRL4	T	CGT	False	8.64386	0.02081	41	2	10.54211	0.08647	41	2	False	L-128
R128(L)V	CGG_naive	R	235	128	L	FWRL4	V	CGT	False	8.58887	-0.03418	28	2	10.49638	0.04073	28	2	False	L-128
R128(L)W	CGG_naive	R	235	128	L	FWRL4	W	CGT	False	8.66728	0.04423	36	2	10.36373	-0.09192	36	2	False	L-128
R128(L)Y	CGG_naive	R	235	128	L	FWRL4	Y	CGT	False	8.63043	0.00737	38	2	10.46643	0.01079	38	2	False	L-128

4400 rows × 19 columns

In [7]:

sns.histplot(data=dms_df, x="delta_bind_CGG")
plt.show()

sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG")
plt.show()

sns.histplot(data=dms_df, x="delta_bind_CGG") plt.show() sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG") plt.show()

In [8]:

dms_df.loc[dms_df.single_nt].delta_bind_CGG.describe()

dms_df.loc[dms_df.single_nt].delta_bind_CGG.describe()

Out[8]:

count    1503.000000
mean       -0.172883
std         0.507691
min        -2.863630
25%        -0.157505
50%         0.000000
75%         0.045450
max         0.877850
Name: delta_bind_CGG, dtype: float64

In [9]:

aa_order = pd.Index(list("RKHDEQNSTYWFAILMVGPC"))
def aa_order_key_fn(aa_index):
    return pd.Index(aa_order.get_loc(aa) for aa in aa_index)

aa_order = pd.Index(list("RKHDEQNSTYWFAILMVGPC")) def aa_order_key_fn(aa_index): return pd.Index(aa_order.get_loc(aa) for aa in aa_index)

In [10]:

dms_df.delta_bind_CGG.describe()

dms_df.delta_bind_CGG.describe()

Out[10]:

count    4397.000000
mean       -0.221130
std         0.540434
min        -3.028910
25%        -0.297030
50%        -0.005230
75%         0.047940
max         0.908430
Name: delta_bind_CGG, dtype: float64

In [11]:

bind_df = dms_df.pivot(index="mutant", columns="site", values="delta_bind_CGG").fillna(0)
accessible_df = dms_df.pivot(index="mutant", columns="site", values="single_nt")
wt_df = dms_df.pivot(index="mutant", columns="site", values="WT")

bind_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True)
accessible_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True)
wt_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True)

annot = pd.DataFrame(np.empty_like(bind_df, dtype=str), index=bind_df.index, columns=bind_df.columns)
annot.iloc[wt_df.values.astype(bool)] = "\u00D7"
# annot.iloc[~accessible_df.values.astype(bool)] = "\u2571"

site_labels_pretty = bind_df.columns.map(lambda x: x.split("-")[0] + str(int(x.split("-")[1])))

plt.figure(figsize=(50, 4))
ax = sns.heatmap(bind_df,
            annot=annot, fmt="", annot_kws=dict(fontweight='bold'),
            cmap=pc.affinity_dms.cmap, norm=pc.affinity_dms.norm,
            cbar_kws=dict(label=r"$-\Delta\log_{10} K_D$"),
            linewidths=0.5, linecolor='grey',
            square=True,
            xticklabels=site_labels_pretty,
)
plt.xlabel("site")
plt.ylabel("amino acid")

ax.collections[0].colorbar.ax.set_yscale('linear')

# Function to draw borders around accessible mutations
def draw_custom_borders(ax, cells):
    for cell in cells:
        i, j = cell
        rect = plt.Rectangle((j, i), 1, 1, fill=False, edgecolor='black', lw=1.5, clip_on=False)
        ax.add_patch(rect)

# Specify accessible cells to have borders
cells_to_border = []
for i, j in zip(*np.where(accessible_df)):
    cells_to_border.append((i, j))
draw_custom_borders(ax, cells_to_border)

plt.tight_layout()
plt.savefig(f"{output_dir}/heatmap.bind.pdf")
plt.show()

bind_df = dms_df.pivot(index="mutant", columns="site", values="delta_bind_CGG").fillna(0) accessible_df = dms_df.pivot(index="mutant", columns="site", values="single_nt") wt_df = dms_df.pivot(index="mutant", columns="site", values="WT") bind_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True) accessible_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True) wt_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True) annot = pd.DataFrame(np.empty_like(bind_df, dtype=str), index=bind_df.index, columns=bind_df.columns) annot.iloc[wt_df.values.astype(bool)] = "\u00D7" # annot.iloc[~accessible_df.values.astype(bool)] = "\u2571" site_labels_pretty = bind_df.columns.map(lambda x: x.split("-")[0] + str(int(x.split("-")[1]))) plt.figure(figsize=(50, 4)) ax = sns.heatmap(bind_df, annot=annot, fmt="", annot_kws=dict(fontweight='bold'), cmap=pc.affinity_dms.cmap, norm=pc.affinity_dms.norm, cbar_kws=dict(label=r"$-\Delta\log_{10} K_D$"), linewidths=0.5, linecolor='grey', square=True, xticklabels=site_labels_pretty, ) plt.xlabel("site") plt.ylabel("amino acid") ax.collections[0].colorbar.ax.set_yscale('linear') # Function to draw borders around accessible mutations def draw_custom_borders(ax, cells): for cell in cells: i, j = cell rect = plt.Rectangle((j, i), 1, 1, fill=False, edgecolor='black', lw=1.5, clip_on=False) ax.add_patch(rect) # Specify accessible cells to have borders cells_to_border = [] for i, j in zip(*np.where(accessible_df)): cells_to_border.append((i, j)) draw_custom_borders(ax, cells_to_border) plt.tight_layout() plt.savefig(f"{output_dir}/heatmap.bind.pdf") plt.show()

In [12]:

sns.histplot(data=dms_df, x="delta_expr")
plt.show()

sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr")
plt.show()

sns.histplot(data=dms_df, x="delta_expr") plt.show() sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr") plt.show()

In [13]:

def make_dms_heatmap(dms_df, observed_var, label, site_labels_pretty, cells_to_border=None, 
                          annot=False, figsize=(50, 4)):
    plot_df = dms_df.pivot(index="mutant", columns="site", values=observed_var).fillna(0)
    plot_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True)

    fig = plt.figure(figsize=figsize)
    
    ax = sns.heatmap(plot_df,
                     annot=annot, fmt="", annot_kws=dict(fontweight='bold'),
                     cmap=pc.expression_dms.cmap, norm=pc.expression_dms.norm,
                     cbar_kws=dict(label=label),
                     linewidths=0.5, linecolor='grey',
                     square=True,
                     xticklabels=site_labels_pretty)

    ax.collections[0].colorbar.ax.set_yscale('linear')

    if cells_to_border is not None:
        draw_custom_borders(ax, cells_to_border)

    plt.xlabel("site")
    plt.ylabel("amino acid")
    plt.tight_layout()

    return fig

fig = make_dms_heatmap(dms_df, 'delta_expr', r"$\Delta$ expression", site_labels_pretty, cells_to_border)
plt.savefig(f"{output_dir}/heatmap.expr.pdf")
plt.show()

def make_dms_heatmap(dms_df, observed_var, label, site_labels_pretty, cells_to_border=None, annot=False, figsize=(50, 4)): plot_df = dms_df.pivot(index="mutant", columns="site", values=observed_var).fillna(0) plot_df.sort_index(key=aa_order_key_fn, axis=0, inplace=True) fig = plt.figure(figsize=figsize) ax = sns.heatmap(plot_df, annot=annot, fmt="", annot_kws=dict(fontweight='bold'), cmap=pc.expression_dms.cmap, norm=pc.expression_dms.norm, cbar_kws=dict(label=label), linewidths=0.5, linecolor='grey', square=True, xticklabels=site_labels_pretty) ax.collections[0].colorbar.ax.set_yscale('linear') if cells_to_border is not None: draw_custom_borders(ax, cells_to_border) plt.xlabel("site") plt.ylabel("amino acid") plt.tight_layout() return fig fig = make_dms_heatmap(dms_df, 'delta_expr', r"$\Delta$ expression", site_labels_pretty, cells_to_border) plt.savefig(f"{output_dir}/heatmap.expr.pdf") plt.show()

Mutations in GC trees¶

Load in metadata

Note that by query'ing the metadata to only inlcude wildtype strain samples, we will not load the LMP2A sample trees and thus ignore them in all downstream analysis. To include these you'll need to reload the metadata and call load_trees again without the query.

In [14]:

metadata = pd.read_csv(metadata_csv, index_col=0) # .query("strain == 'wt'")
metadata.head()

metadata = pd.read_csv(metadata_csv, index_col=0) # .query("strain == 'wt'") metadata.head()

Out[14]:

	ngs_id	imm_duration	mouse	gc	strain	node	cell_type	plate	hc_barcode	lc_barcode	row	col
uid
D15_M1_GC1	PR-2-01	d15	1	1	wt	RP	GC	2	9	9	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M1_GC2	PR-2-01	d15	1	2	wt	RI	GC	3	2	1	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M1_GC3	PR-2-01	d15	1	3	wt	LI	GC	4	14	2	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M2_GC4	PR-2-01	d15	2	4	wt	RP	GC	5	10	11	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M3_GC5	PR-2-01	d15	3	5	wt	RP	GC	6	7	4	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12

Load in trees

In [15]:

trees = ut.load_trees(metadata, results, ranking_subdir)

trees = ut.load_trees(metadata, results, ranking_subdir)

missing tree for D20_M28_GC131
missing tree for D20_M28_GC135

GC metadata

In [16]:

sns.histplot(data=metadata, x="imm_duration")
plt.show()
sns.histplot(data=metadata, x="cell_type")
plt.show()
sns.histplot(data=metadata, x="node")
plt.show()
sns.histplot(data=metadata, x="strain")
plt.show()

metadata.to_csv(f"{output_dir}/metadata.renamed.csv")
metadata

sns.histplot(data=metadata, x="imm_duration") plt.show() sns.histplot(data=metadata, x="cell_type") plt.show() sns.histplot(data=metadata, x="node") plt.show() sns.histplot(data=metadata, x="strain") plt.show() metadata.to_csv(f"{output_dir}/metadata.renamed.csv") metadata

Out[16]:

	ngs_id	imm_duration	mouse	gc	strain	node	cell_type	plate	hc_barcode	lc_barcode	row	col
uid
D15_M1_GC1	PR-2-01	d15	1	1	wt	RP	GC	2	9	9	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M1_GC2	PR-2-01	d15	1	2	wt	RI	GC	3	2	1	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M1_GC3	PR-2-01	d15	1	3	wt	LI	GC	4	14	2	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M2_GC4	PR-2-01	d15	2	4	wt	RP	GC	5	10	11	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D15_M3_GC5	PR-2-01	d15	3	5	wt	RP	GC	6	7	4	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
...	...	...	...	...	...	...	...	...	...	...	...	...
D20_M29_GC145	PR-1-10	d20	29	145	LMP2A	RB	GC	126	6	6	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D20_M29_GC146	PR-1-10	d20	29	146	LMP2A	RB	GC	127	7	7	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D20_M29_GC147	PR-1-10	d20	29	147	LMP2A	LB	GC	128	8	8	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D20_M29_GC148	PR-1-10	d20	29	148	LMP2A	LB	GC	129	9	9	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12
D20_M29_GC149	PR-1-10	d20	29	149	LMP2A	LA	GC	130	10	10	A.B.C.D.E.F.G.H	1.2.3.4.5.6.7.8.9.10.11.12

149 rows × 12 columns

In [17]:

pos_df = pd.read_csv(dms_sites, dtype=dict(site=pd.Int16Dtype()), index_col="site_scFv")
pos_df

pos_df = pd.read_csv(dms_sites, dtype=dict(site=pd.Int16Dtype()), index_col="site_scFv") pos_df

Out[17]:

	amino_acid	chain	site	KI_codon	annotation
site_scFv
1	E	H	1	GAG	FWRH1
2	V	H	2	GTG	FWRH1
3	Q	H	3	CAG	FWRH1
4	L	H	4	CTT	FWRH1
5	Q	H	5	CAG	FWRH1
...	...	...	...	...	...
231	L	L	124	CTA	FWRL4
232	E	L	125	GAA	FWRL4
233	I	L	126	ATA	FWRL4
234	K	L	127	AAA	FWRL4
235	R	L	128	CGT	FWRL4

235 rows × 5 columns

In [18]:

dms_df

dms_df

Out[18]:

	target	wildtype	position	position_IMGT	chain	annotation	mutant	codon	single_nt	bind_CGG	delta_bind_CGG	n_bc_bind_CGG	n_libs_bind_CGG	expr	delta_expr	n_bc_expr	n_libs_expr	WT	site
mutation
E1(H)A	CGG_naive	E	1	1	H	FWRH1	A	GAG	True	8.74239	0.11933	20	2	10.45318	-0.00246	20	2	False	H-001
E1(H)C	CGG_naive	E	1	1	H	FWRH1	C	GAG	False	8.60813	-0.01492	22	2	10.33348	-0.12216	22	2	False	H-001
E1(H)D	CGG_naive	E	1	1	H	FWRH1	D	GAG	True	8.63554	0.01249	18	2	10.50438	0.04874	18	2	False	H-001
E1(H)E	CGG_naive	E	1	1	H	FWRH1	E	GAG	True	8.62305	0.00000	23285	2	10.45565	0.00000	23285	2	True	H-001
E1(H)F	CGG_naive	E	1	1	H	FWRH1	F	GAG	False	8.75738	0.13433	29	2	10.34185	-0.11379	29	2	False	H-001
...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...
R128(L)S	CGG_naive	R	235	128	L	FWRL4	S	CGT	True	8.63400	0.01095	45	2	10.52908	0.07344	45	2	False	L-128
R128(L)T	CGG_naive	R	235	128	L	FWRL4	T	CGT	False	8.64386	0.02081	41	2	10.54211	0.08647	41	2	False	L-128
R128(L)V	CGG_naive	R	235	128	L	FWRL4	V	CGT	False	8.58887	-0.03418	28	2	10.49638	0.04073	28	2	False	L-128
R128(L)W	CGG_naive	R	235	128	L	FWRL4	W	CGT	False	8.66728	0.04423	36	2	10.36373	-0.09192	36	2	False	L-128
R128(L)Y	CGG_naive	R	235	128	L	FWRL4	Y	CGT	False	8.63043	0.00737	38	2	10.46643	0.01079	38	2	False	L-128

4400 rows × 19 columns

In [19]:

igh_pos_map = pos_df.loc[pos_df.chain == "H", "site"].reset_index(drop=True)
igk_pos_map = pos_df.loc[pos_df.chain == "L", "site"].reset_index(drop=True)

igh_pos_map = pos_df.loc[pos_df.chain == "H", "site"].reset_index(drop=True) igk_pos_map = pos_df.loc[pos_df.chain == "L", "site"].reset_index(drop=True)

Mutation events¶

In [20]:

hits_df = pd.DataFrame(np.zeros_like(bind_df.values), index=bind_df.index, columns=bind_df.columns)

experiments = ["15-day", "20-day", "10-week", "LMP2A"]
suffixes = ["events", "abundance"]
for experiment, suffix in itertools.product(experiments, suffixes):
    dms_df[f"mutation {suffix} ({experiment})"] = 0

total_abundance_dict = {experiment: 0 for experiment in experiments}

for gc, tree in trees.items():
    if gc not in metadata.index:
        print(f"WARNING: {gc} not found in metadata")
        continue
    if (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "d15"):
        suffix = "15-day"
    elif (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "d20"):
        suffix = "20-day"
    elif (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "w10"):
        suffix = "10-week"
    elif (metadata.strain[gc] == "LMP2A"):
        suffix = "LMP2A"
    else:
        raise ValueError(f"unknown gc {gc}")
    for node in tree.tree.iter_descendants(strategy="preorder"):
        total_abundance_dict[suffix] += node.abundance
        # Loop over new mutations.
        for mutation in set(node.mutations) - set(node.up.mutations):
            mutant = mutation[-1]
            if mutant != "*":
                site = dms_df.site[mutation]
                hits_df.loc[mutant, site] += 1
                dms_df.loc[mutation, f"mutation events ({suffix})"] += 1
        # Loop over all mutations.
        for mutation in node.mutations:
            mutant = mutation[-1]
            if mutant != "*":
                dms_df.loc[mutation, f"mutation abundance ({suffix})"] += node.abundance

# NOTE: overall mutation abundance means day 15 and day 20
for suffix in suffixes:
    dms_df[f"mutation {suffix}"] = dms_df[f"mutation {suffix} (15-day)"] + dms_df[f"mutation {suffix} (20-day)"]

hits_df

hits_df = pd.DataFrame(np.zeros_like(bind_df.values), index=bind_df.index, columns=bind_df.columns) experiments = ["15-day", "20-day", "10-week", "LMP2A"] suffixes = ["events", "abundance"] for experiment, suffix in itertools.product(experiments, suffixes): dms_df[f"mutation {suffix} ({experiment})"] = 0 total_abundance_dict = {experiment: 0 for experiment in experiments} for gc, tree in trees.items(): if gc not in metadata.index: print(f"WARNING: {gc} not found in metadata") continue if (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "d15"): suffix = "15-day" elif (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "d20"): suffix = "20-day" elif (metadata.strain[gc] == "wt") and (metadata.imm_duration[gc] == "w10"): suffix = "10-week" elif (metadata.strain[gc] == "LMP2A"): suffix = "LMP2A" else: raise ValueError(f"unknown gc {gc}") for node in tree.tree.iter_descendants(strategy="preorder"): total_abundance_dict[suffix] += node.abundance # Loop over new mutations. for mutation in set(node.mutations) - set(node.up.mutations): mutant = mutation[-1] if mutant != "*": site = dms_df.site[mutation] hits_df.loc[mutant, site] += 1 dms_df.loc[mutation, f"mutation events ({suffix})"] += 1 # Loop over all mutations. for mutation in node.mutations: mutant = mutation[-1] if mutant != "*": dms_df.loc[mutation, f"mutation abundance ({suffix})"] += node.abundance # NOTE: overall mutation abundance means day 15 and day 20 for suffix in suffixes: dms_df[f"mutation {suffix}"] = dms_df[f"mutation {suffix} (15-day)"] + dms_df[f"mutation {suffix} (20-day)"] hits_df

Out[20]:

site	H-001	H-002	H-003	H-004	H-005	H-006	H-007	H-008	H-009	H-011	...	L-119	L-120	L-121	L-122	L-123	L-124	L-125	L-126	L-127	L-128
mutant
R	0.0	0.0	13.0	0.0	4.0	0.0	0.0	2.0	1.0	8.0	...	0.0	0.0	1.0	0.0	30.0	0.0	0.0	18.0	62.0	0.0
K	2.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	0.0	0.0	0.0	19.0	27.0	0.0	0.0
H	0.0	0.0	10.0	1.0	1.0	0.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	0.0	2.0	0.0	1.0	0.0	0.0	0.0
D	17.0	0.0	0.0	0.0	0.0	1.0	0.0	1.0	0.0	0.0	...	5.0	0.0	0.0	0.0	4.0	0.0	39.0	0.0	0.0	0.0
E	0.0	0.0	1.0	0.0	0.0	0.0	0.0	1.0	0.0	0.0	...	0.0	0.0	9.0	0.0	51.0	0.0	0.0	0.0	40.0	0.0
Q	1.0	0.0	0.0	0.0	0.0	1.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	0.0	53.0	5.0	33.0	0.0	28.0	0.0
N	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	10.0	...	0.0	0.0	0.0	0.0	133.0	0.0	1.0	1.0	25.0	0.0
S	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	12.0	1.0	2.0	0.0	0.0	3.0	0.0
T	0.0	0.0	0.0	0.0	0.0	0.0	1.0	0.0	0.0	8.0	...	0.0	2.0	0.0	0.0	21.0	0.0	0.0	24.0	14.0	0.0
Y	1.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	2.0	...	0.0	0.0	0.0	0.0	2.0	0.0	0.0	0.0	1.0	0.0
W	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	...	0.0	2.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
F	0.0	0.0	0.0	14.0	0.0	0.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	0.0	0.0	1.0	0.0	1.0	0.0	0.0
A	6.0	4.0	0.0	0.0	0.0	11.0	2.0	0.0	0.0	0.0	...	8.0	13.0	0.0	4.0	1.0	1.0	8.0	2.0	0.0	0.0
I	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	2.0	...	0.0	0.0	0.0	6.0	1.0	3.0	0.0	0.0	20.0	0.0
L	0.0	0.0	3.0	0.0	0.0	0.0	1.0	0.0	2.0	0.0	...	0.0	2.0	0.0	0.0	1.0	0.0	0.0	43.0	1.0	0.0
M	0.0	3.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	...	0.0	0.0	0.0	0.0	9.0	0.0	0.0	93.0	1.0	0.0
V	2.0	0.0	0.0	4.0	0.0	0.0	2.0	0.0	0.0	0.0	...	0.0	0.0	6.0	1.0	2.0	171.0	1.0	55.0	0.0	0.0
G	9.0	2.0	0.0	0.0	0.0	14.0	0.0	0.0	0.0	15.0	...	0.0	3.0	0.0	0.0	1.0	0.0	2.0	0.0	1.0	0.0
P	0.0	0.0	1.0	13.0	1.0	0.0	0.0	0.0	0.0	0.0	...	0.0	8.0	0.0	0.0	0.0	4.0	0.0	1.0	0.0	0.0
C	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	7.0	...	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0

20 rows × 220 columns

In [21]:

plt.figure(figsize=(30, 8))

plt.subplot(211)
g = sns.stripplot(data=hits_df)
for item in g.get_xticklabels():
    item.set_rotation(90)

plt.subplot(212, sharex=g)
g = sns.barplot(data=hits_df, errorbar=None, estimator=sum)
for item in g.get_xticklabels():
    item.set_rotation(90)

plt.tight_layout()
plt.show()

plt.figure(figsize=(30, 8)) plt.subplot(211) g = sns.stripplot(data=hits_df) for item in g.get_xticklabels(): item.set_rotation(90) plt.subplot(212, sharex=g) g = sns.barplot(data=hits_df, errorbar=None, estimator=sum) for item in g.get_xticklabels(): item.set_rotation(90) plt.tight_layout() plt.show()

In [22]:

plt.figure(figsize=(50, 4))
sns.heatmap(hits_df, annot=annot, fmt="", annot_kws=dict(fontweight="bold"),
            cmap="Reds",
#             vmin=100,
            vmax=200,
            cbar_kws=dict(label="number of mutations in GC trees"), linewidths=.1, square=True)
plt.xlabel("site")
plt.ylabel("amino acid")
plt.show()

plt.figure(figsize=(50, 4)) sns.heatmap(hits_df, annot=annot, fmt="", annot_kws=dict(fontweight="bold"), cmap="Reds", # vmin=100, vmax=200, cbar_kws=dict(label="number of mutations in GC trees"), linewidths=.1, square=True) plt.xlabel("site") plt.ylabel("amino acid") plt.show()

Add mutability to dms_df¶

In [23]:

from math import isnan
from Bio.codonalign.codonseq import CodonSeq


chigy_hc_passenger = pd.read_csv(chigy_hc_mut_rates, index_col=0)
chigy_lc_passenger = pd.read_csv(chigy_lc_mut_rates, index_col=0)

mutation_rates_nt = pd.concat([chigy_hc_passenger.head(-1), chigy_lc_passenger.head(-1)])
# Assert that we've glued things together covering all sites
assert (mutation_rates_nt.index == np.arange(len(mutation_rates_nt))).all()


# mutability = pd.read_csv("../nextflow/data/mutability/MK_RS5NF_mutability.csv", sep=" ", index_col=0).squeeze("columns")
# substitution = pd.read_csv("../nextflow/data/mutability/MK_RS5NF_substitution.csv", sep=" ", index_col=0)

naive = next(iter(trees.values())).tree.sequence
igk_idx = 336

# padded_igh = "NN" + naive[:igk_idx] + "NN"
# padded_igk = "NN" + naive[igk_idx:] + "NN"
# # mutabilities of each nucleotide
# contexts = [padded_igh[(i - 2) : (i + 3)]
#                 for i in range(2, len(padded_igh) - 2)
#                ] + [padded_igk[(i - 2) : (i + 3)]
#                     for i in range(2, len(padded_igk) - 2)
#                    ]

# # mutation rates at each site in naive
# mutation_rates_nt = pd.concat((mutability[context] * substitution.loc[context] for context in contexts), axis=1).T.reset_index()

# now we want the above at the codon level

codons = CodonSeq(naive)

# assert len(contexts) == len(naive) == len(codons)

dms_df["mutation rate"] = np.nan

for codon_idx in range(codons.get_codon_num()):
    codon = CodonSeq(codons.get_codon(codon_idx))
    aa = codon.translate()
    for codon_pos in range(3):
        idx = 3 * codon_idx + codon_pos
        if idx < igk_idx:
            chain = "H"
            imgt_idx = igh_pos_map[idx // 3]
        else:
            chain = "L"
            imgt_idx = igk_pos_map[(idx - igk_idx) // 3]
        assert naive[idx] == codon[codon_pos]
        for alt_nt in "ACGT":
            if alt_nt != naive[idx]:
                mutated_codon = list(codon)
                mutated_codon[codon_pos] = alt_nt
                mutated_codon = CodonSeq("".join(mutated_codon))
                mutated_aa = mutated_codon.translate()
                if mutated_aa != "*" and mutated_aa != aa:
                    mutation = f"{aa}{imgt_idx}({chain}){mutated_aa}"
                    if np.isnan(dms_df.loc[mutation, "mutation rate"]):
                        dms_df.loc[mutation, "mutation rate"] = 0
                    dms_df.loc[mutation, "mutation rate"] += mutation_rates_nt.loc[idx, alt_nt]
dms_df

from math import isnan from Bio.codonalign.codonseq import CodonSeq chigy_hc_passenger = pd.read_csv(chigy_hc_mut_rates, index_col=0) chigy_lc_passenger = pd.read_csv(chigy_lc_mut_rates, index_col=0) mutation_rates_nt = pd.concat([chigy_hc_passenger.head(-1), chigy_lc_passenger.head(-1)]) # Assert that we've glued things together covering all sites assert (mutation_rates_nt.index == np.arange(len(mutation_rates_nt))).all() # mutability = pd.read_csv("../nextflow/data/mutability/MK_RS5NF_mutability.csv", sep=" ", index_col=0).squeeze("columns") # substitution = pd.read_csv("../nextflow/data/mutability/MK_RS5NF_substitution.csv", sep=" ", index_col=0) naive = next(iter(trees.values())).tree.sequence igk_idx = 336 # padded_igh = "NN" + naive[:igk_idx] + "NN" # padded_igk = "NN" + naive[igk_idx:] + "NN" # # mutabilities of each nucleotide # contexts = [padded_igh[(i - 2) : (i + 3)] # for i in range(2, len(padded_igh) - 2) # ] + [padded_igk[(i - 2) : (i + 3)] # for i in range(2, len(padded_igk) - 2) # ] # # mutation rates at each site in naive # mutation_rates_nt = pd.concat((mutability[context] * substitution.loc[context] for context in contexts), axis=1).T.reset_index() # now we want the above at the codon level codons = CodonSeq(naive) # assert len(contexts) == len(naive) == len(codons) dms_df["mutation rate"] = np.nan for codon_idx in range(codons.get_codon_num()): codon = CodonSeq(codons.get_codon(codon_idx)) aa = codon.translate() for codon_pos in range(3): idx = 3 * codon_idx + codon_pos if idx < igk_idx: chain = "H" imgt_idx = igh_pos_map[idx // 3] else: chain = "L" imgt_idx = igk_pos_map[(idx - igk_idx) // 3] assert naive[idx] == codon[codon_pos] for alt_nt in "ACGT": if alt_nt != naive[idx]: mutated_codon = list(codon) mutated_codon[codon_pos] = alt_nt mutated_codon = CodonSeq("".join(mutated_codon)) mutated_aa = mutated_codon.translate() if mutated_aa != "*" and mutated_aa != aa: mutation = f"{aa}{imgt_idx}({chain}){mutated_aa}" if np.isnan(dms_df.loc[mutation, "mutation rate"]): dms_df.loc[mutation, "mutation rate"] = 0 dms_df.loc[mutation, "mutation rate"] += mutation_rates_nt.loc[idx, alt_nt] dms_df

/opt/conda/envs/replay/lib/python3.10/site-packages/Bio/codonalign/__init__.py:21: BiopythonExperimentalWarning: Bio.codonalign is an experimental module which may undergo significant changes prior to its future official release.
  warnings.warn(

Out[23]:

	target	wildtype	position	position_IMGT	chain	annotation	mutant	codon	single_nt	bind_CGG	...	mutation abundance (15-day)	mutation events (20-day)	mutation abundance (20-day)	mutation events (10-week)	mutation abundance (10-week)	mutation events (LMP2A)	mutation abundance (LMP2A)	mutation events	mutation abundance	mutation rate
mutation
E1(H)A	CGG_naive	E	1	1	H	FWRH1	A	GAG	True	8.74239	...	2	4	4	0	0	0	0	6	6	0.000006
E1(H)C	CGG_naive	E	1	1	H	FWRH1	C	GAG	False	8.60813	...	0	0	0	0	0	0	0	0	0	NaN
E1(H)D	CGG_naive	E	1	1	H	FWRH1	D	GAG	True	8.63554	...	3	7	54	5	8	2	4	10	57	0.000725
E1(H)E	CGG_naive	E	1	1	H	FWRH1	E	GAG	True	8.62305	...	0	0	0	0	0	0	0	0	0	NaN
E1(H)F	CGG_naive	E	1	1	H	FWRH1	F	GAG	False	8.75738	...	0	0	0	0	0	0	0	0	0	NaN
...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...
R128(L)S	CGG_naive	R	235	128	L	FWRL4	S	CGT	True	8.63400	...	0	0	0	0	0	0	0	0	0	NaN
R128(L)T	CGG_naive	R	235	128	L	FWRL4	T	CGT	False	8.64386	...	0	0	0	0	0	0	0	0	0	NaN
R128(L)V	CGG_naive	R	235	128	L	FWRL4	V	CGT	False	8.58887	...	0	0	0	0	0	0	0	0	0	NaN
R128(L)W	CGG_naive	R	235	128	L	FWRL4	W	CGT	False	8.66728	...	0	0	0	0	0	0	0	0	0	NaN
R128(L)Y	CGG_naive	R	235	128	L	FWRL4	Y	CGT	False	8.63043	...	0	0	0	0	0	0	0	0	0	NaN

4400 rows × 30 columns

In [24]:

assert (~dms_df.single_nt & (dms_df["mutation rate"] > 0)).sum() == 0

assert (~dms_df.single_nt & (dms_df["mutation rate"] > 0)).sum() == 0

In [25]:

sns.histplot(data=dms_df.query("single_nt == True"), x="delta_bind_CGG", bins=50, stat="density")
sns.histplot(data=dms_df.query("single_nt == True"), x="delta_bind_CGG", weights="mutation rate", bins=50, stat="density")

sns.histplot(data=dms_df.query("single_nt == True"), x="delta_bind_CGG", bins=50, stat="density") sns.histplot(data=dms_df.query("single_nt == True"), x="delta_bind_CGG", weights="mutation rate", bins=50, stat="density")

Out[25]:

<Axes: xlabel='delta_bind_CGG', ylabel='Density'>

In [26]:

dms_df.loc[(dms_df["mutation events"]>100)].sort_values("mutation events")[["delta_bind_CGG", "wildtype", "mutant", "mutation events", "mutation rate"]]

dms_df.loc[(dms_df["mutation events"]>100)].sort_values("mutation events")[["delta_bind_CGG", "wildtype", "mutant", "mutation events", "mutation rate"]]

Out[26]:

	delta_bind_CGG	wildtype	mutant	mutation events	mutation rate
mutation
Q86(H)H	-0.00100	Q	H	101	0.004546
Y88(H)F	0.05302	Y	F	105	0.005637
N92(H)S	-0.09063	N	S	106	0.006922
N108(L)Y	0.73212	N	Y	106	0.004310
K123(L)N	-0.04793	K	N	109	0.008890
L50(H)F	-0.00532	L	F	111	0.004655
S109(L)T	0.01760	S	T	119	0.007761
G36(L)D	-0.59849	G	D	122	0.010422
Y103(H)F	0.13457	Y	F	123	0.008410
S92(L)N	0.03607	S	N	126	0.006265
A40(L)G	0.80414	A	G	132	0.000699
T96(H)S	0.04358	T	S	134	0.008485
R67(L)K	0.15471	R	K	136	0.003038
L124(L)V	0.08206	L	V	142	0.010172
S64(H)T	-0.02745	S	T	144	0.009512
S64(H)R	0.87785	S	R	171	0.004167
Y42(L)F	-0.03927	Y	F	172	0.007922
E101(L)D	-0.11838	E	D	181	0.012170
S109(L)R	0.79500	S	R	183	0.007756
A105(H)G	0.44629	A	G	195	0.001208
S109(L)N	0.01231	S	N	214	0.015845
Q105(L)H	0.38030	Q	H	214	0.007117
N108(L)K	0.85965	N	K	245	0.003729
S64(H)N	0.05093	S	N	294	0.019506
S57(H)N	0.12761	S	N	424	0.022500

In [27]:

pos_df.loc[90:100]

pos_df.loc[90:100]

Out[27]:

	amino_acid	chain	site	KI_codon	annotation
site_scFv
90	T	H	99	ACA	FWRH3
91	A	H	100	GCC	FWRH3
92	T	H	101	ACA	FWRH3
93	Y	H	102	TAT	FWRH3
94	Y	H	103	TAC	FWRH3
95	C	H	104	TGT	FWRH3
96	A	H	105	GCA	CDRH3
97	R	H	106	AGG	CDRH3
98	D	H	107	GAC	CDRH3
99	F	H	115	TTC	CDRH3
100	D	H	116	GAT	CDRH3

In [28]:

dms_df.columns

dms_df.columns

Out[28]:

Index(['target', 'wildtype', 'position', 'position_IMGT', 'chain',
       'annotation', 'mutant', 'codon', 'single_nt', 'bind_CGG',
       'delta_bind_CGG', 'n_bc_bind_CGG', 'n_libs_bind_CGG', 'expr',
       'delta_expr', 'n_bc_expr', 'n_libs_expr', 'WT', 'site',
       'mutation events (15-day)', 'mutation abundance (15-day)',
       'mutation events (20-day)', 'mutation abundance (20-day)',
       'mutation events (10-week)', 'mutation abundance (10-week)',
       'mutation events (LMP2A)', 'mutation abundance (LMP2A)',
       'mutation events', 'mutation abundance', 'mutation rate'],
      dtype='object')

In [29]:

query_str = "(single_nt == True) & (WT == False)"
# Slope is ML estimator for Poisson regression using an identity link function.
# Here we set this up for the complete dataset but also use the same formula for the per-timepoint data below.

for suffix in ["", " (15-day)", " (20-day)"]:
    abundance_key = f"mutation abundance{suffix}"
    slope = dms_df.query(query_str)[abundance_key].sum() / dms_df.query(query_str)["mutation rate"].sum()
    rate_grid = np.geomspace(1e-4, dms_df.query(query_str)["mutation rate"].max(), 100)
    predicted_events = slope * dms_df.query(query_str)["mutation rate"]
    dms_df["mutation enrichment"+suffix] = np.log10((.5 + dms_df.query(query_str)[abundance_key]) / (.5 + predicted_events))

# Scale the LMP2A data to accomadate the difference in total cellular abundance between the different experiments.
lmp2a_predicted_events = predicted_events * total_abundance_dict["LMP2A"] / (total_abundance_dict["15-day"] + total_abundance_dict["20-day"])
dms_df["mutation enrichment (LMP2A)"] = np.log10((.5 + dms_df.query(query_str)["mutation abundance (LMP2A)"]) / (.5 + lmp2a_predicted_events))

query_str = "(single_nt == True) & (WT == False)" # Slope is ML estimator for Poisson regression using an identity link function. # Here we set this up for the complete dataset but also use the same formula for the per-timepoint data below. for suffix in ["", " (15-day)", " (20-day)"]: abundance_key = f"mutation abundance{suffix}" slope = dms_df.query(query_str)[abundance_key].sum() / dms_df.query(query_str)["mutation rate"].sum() rate_grid = np.geomspace(1e-4, dms_df.query(query_str)["mutation rate"].max(), 100) predicted_events = slope * dms_df.query(query_str)["mutation rate"] dms_df["mutation enrichment"+suffix] = np.log10((.5 + dms_df.query(query_str)[abundance_key]) / (.5 + predicted_events)) # Scale the LMP2A data to accomadate the difference in total cellular abundance between the different experiments. lmp2a_predicted_events = predicted_events * total_abundance_dict["LMP2A"] / (total_abundance_dict["15-day"] + total_abundance_dict["20-day"]) dms_df["mutation enrichment (LMP2A)"] = np.log10((.5 + dms_df.query(query_str)["mutation abundance (LMP2A)"]) / (.5 + lmp2a_predicted_events))

In [30]:

def plot_one_measured_var(axs, dms_df, query_str, dependent_var, measured_var, cmap, norm, linthresh=1, linscale=0.2):
    """
    Generate a row of plots for a given measured variable.
    
    Args:
        axs (array): Array of matplotlib axes to fill.
        dms_df (pd.DataFrame): Dataframe containing the data.
        query_str (str): Query string to filter the data.
        dependent_var (str): Name of the dependent variable column.
        measured_var (str): Name of the measured variable (e.g., "affinity" or "expression").
        bins (list): Bin edges for the point plots.
        cmap: Colormap for scatter plots.
        norm: Normalization for colormap.
        linthresh (float): Linthresh for symlog scaling.
        linscale (float): Linscale for symlog scaling.
    """
    data = dms_df.query(query_str).sort_values(by=f"delta_{measured_var}", key=lambda val: np.abs(val)).dropna()
    max_y = data[dependent_var].max() * 1.5
    min_y = -1


    # Left plot: Mutation abundance vs mutation rate
    ax0 = axs[0]
    scatter = ax0.scatter(
        data["mutation rate"], data[dependent_var],
        c=data[f"delta_{measured_var}"], s=13, alpha=0.8, cmap=cmap, norm=norm,
        clip_on=False, edgecolors="k", linewidths=0.1
    )
    plt.colorbar(scatter, ax=ax0, label=fr"$\Delta$ {measured_var}")
    ax0.collections[0].colorbar.ax.set_yscale('linear')
    ax0.set_xscale("log")
    ax0.set_yscale("symlog", linthresh=linthresh, linscale=linscale)
    ax0.set_xlabel("mutation rate")
    ax0.set_ylabel(dependent_var)
    ax0.set_ylim(top=max_y, bottom=min_y)

    log_mutation_rate = np.log10(data["mutation rate"])
    y0 = data[dependent_var]

    slope = data[dependent_var].sum() / data["mutation rate"].sum()
    rate_grid = np.geomspace(1e-4, data["mutation rate"].max(), 100)
    ax0.plot(rate_grid, slope*rate_grid, color="k", lw=1)
    
    # Compute Spearman's rho for log mutation rate vs dependent variable
    rho0, _ = spearmanr(log_mutation_rate, y0)
    print(f"Spearman's ρ for log(mutation rate) vs {dependent_var}: {rho0:.4f}")

    # Middle plot: Delta affinity vs mutation abundance
    ax1 = axs[1]
    scatter = ax1.scatter(
        data[f"delta_{measured_var}"],
        data[dependent_var],
        s=6.5,
        alpha=0.5,
        c=np.log10(data["mutation rate"]),
        cmap="viridis",
        clip_on=False,
    )
    cbar = plt.colorbar(scatter, ax=ax1)
    cbar.set_label("$\log_{10}$ mutation rate")

    ax1.axhline(0, color="k", linestyle="--", linewidth=1)
    ax1.axvline(0, color="k", linestyle="--", linewidth=1)
    ax1.set_xlabel(fr"$\Delta$ {measured_var}")
    ax1.set_ylabel(dependent_var)
    ax1.set_yscale("symlog", linthresh=linthresh, linscale=linscale)
    ax1.set_ylim(top=max_y, bottom=min_y)

    x1 = data[f"delta_{measured_var}"]
    y1 = data[dependent_var]

    rho1, _ = spearmanr(x1, y1)
    print(f"Spearman's ρ for Δ{measured_var} vs {dependent_var}: {rho1:.4f}")
        
    enrichment_var = re.sub("abundance", "enrichment", dependent_var)
    # Right plot: Mutation enrichment lowess
    predicted_events = slope * data["mutation rate"]
    data[enrichment_var] = np.log10((0.5 + data[dependent_var]) / (0.5 + predicted_events))
    ax2 = axs[2]
    regplot_lowess_ci(data=data,
            x=f"delta_{measured_var}", y=enrichment_var,
            ci_level=95, n_boot=100,
            scatter_kwargs=dict(s=13, alpha=0.5, color="grey"),
            line_kwargs=dict(color="xkcd:orange"),
            ax=ax2,
            )
    ax2.axhline(0, color="k", linestyle="--", linewidth=1)
    ax2.axvline(0, color="k", linestyle="--", linewidth=1)
    ax2.set_xlabel(fr"$\Delta$ {measured_var}")
    ax2.set_ylabel(enrichment_var)
    

def mutations_plot(dms_df, timepoint=""):
    """
    Generate a 2x3 plot layout with rows for affinity and expression.
    
    Args:
        dms_df (pd.DataFrame): Dataframe containing the data.
        dependent_var (str): Name of the dependent variable column.
        output_dir (str): Output directory for saving the plot.
    """

    dms_df = dms_df.copy()
    dms_df.rename(columns={"delta_bind_CGG": "delta_affinity"}, inplace=True)
    dms_df.rename(columns={"delta_expr": "delta_expression"}, inplace=True)

    if timepoint == "":
        dependent_var = "mutation abundance"
    else:
        dependent_var = f"mutation abundance ({timepoint})"

    query_str="(single_nt == True) & (WT == False)"
    
    plt.figure(figsize=(13, 7))
    axs = [plt.subplot(231), plt.subplot(232), plt.subplot(233)]
    plot_one_measured_var(
        axs, dms_df, query_str, dependent_var, "affinity",
        cmap=pc.affinity_dms.cmap, norm=pc.affinity_dms.norm
    )
    
    axs = [plt.subplot(234), plt.subplot(235), plt.subplot(236)]
    plot_one_measured_var(
        axs, dms_df, query_str, dependent_var, "expression",
        cmap=pc.expression_dms.cmap, norm=pc.expression_dms.norm
    )
    
    if timepoint != "":
        plt.suptitle(f"{timepoint}")
    plt.tight_layout()
    plt.savefig(f"{output_dir}/eventsVsrate{timepoint}.pdf")
    plt.show()

def plot_one_measured_var(axs, dms_df, query_str, dependent_var, measured_var, cmap, norm, linthresh=1, linscale=0.2): """ Generate a row of plots for a given measured variable. Args: axs (array): Array of matplotlib axes to fill. dms_df (pd.DataFrame): Dataframe containing the data. query_str (str): Query string to filter the data. dependent_var (str): Name of the dependent variable column. measured_var (str): Name of the measured variable (e.g., "affinity" or "expression"). bins (list): Bin edges for the point plots. cmap: Colormap for scatter plots. norm: Normalization for colormap. linthresh (float): Linthresh for symlog scaling. linscale (float): Linscale for symlog scaling. """ data = dms_df.query(query_str).sort_values(by=f"delta_{measured_var}", key=lambda val: np.abs(val)).dropna() max_y = data[dependent_var].max() * 1.5 min_y = -1 # Left plot: Mutation abundance vs mutation rate ax0 = axs[0] scatter = ax0.scatter( data["mutation rate"], data[dependent_var], c=data[f"delta_{measured_var}"], s=13, alpha=0.8, cmap=cmap, norm=norm, clip_on=False, edgecolors="k", linewidths=0.1 ) plt.colorbar(scatter, ax=ax0, label=fr"$\Delta$ {measured_var}") ax0.collections[0].colorbar.ax.set_yscale('linear') ax0.set_xscale("log") ax0.set_yscale("symlog", linthresh=linthresh, linscale=linscale) ax0.set_xlabel("mutation rate") ax0.set_ylabel(dependent_var) ax0.set_ylim(top=max_y, bottom=min_y) log_mutation_rate = np.log10(data["mutation rate"]) y0 = data[dependent_var] slope = data[dependent_var].sum() / data["mutation rate"].sum() rate_grid = np.geomspace(1e-4, data["mutation rate"].max(), 100) ax0.plot(rate_grid, slope*rate_grid, color="k", lw=1) # Compute Spearman's rho for log mutation rate vs dependent variable rho0, _ = spearmanr(log_mutation_rate, y0) print(f"Spearman's ρ for log(mutation rate) vs {dependent_var}: {rho0:.4f}") # Middle plot: Delta affinity vs mutation abundance ax1 = axs[1] scatter = ax1.scatter( data[f"delta_{measured_var}"], data[dependent_var], s=6.5, alpha=0.5, c=np.log10(data["mutation rate"]), cmap="viridis", clip_on=False, ) cbar = plt.colorbar(scatter, ax=ax1) cbar.set_label("$\log_{10}$ mutation rate") ax1.axhline(0, color="k", linestyle="--", linewidth=1) ax1.axvline(0, color="k", linestyle="--", linewidth=1) ax1.set_xlabel(fr"$\Delta$ {measured_var}") ax1.set_ylabel(dependent_var) ax1.set_yscale("symlog", linthresh=linthresh, linscale=linscale) ax1.set_ylim(top=max_y, bottom=min_y) x1 = data[f"delta_{measured_var}"] y1 = data[dependent_var] rho1, _ = spearmanr(x1, y1) print(f"Spearman's ρ for Δ{measured_var} vs {dependent_var}: {rho1:.4f}") enrichment_var = re.sub("abundance", "enrichment", dependent_var) # Right plot: Mutation enrichment lowess predicted_events = slope * data["mutation rate"] data[enrichment_var] = np.log10((0.5 + data[dependent_var]) / (0.5 + predicted_events)) ax2 = axs[2] regplot_lowess_ci(data=data, x=f"delta_{measured_var}", y=enrichment_var, ci_level=95, n_boot=100, scatter_kwargs=dict(s=13, alpha=0.5, color="grey"), line_kwargs=dict(color="xkcd:orange"), ax=ax2, ) ax2.axhline(0, color="k", linestyle="--", linewidth=1) ax2.axvline(0, color="k", linestyle="--", linewidth=1) ax2.set_xlabel(fr"$\Delta$ {measured_var}") ax2.set_ylabel(enrichment_var) def mutations_plot(dms_df, timepoint=""): """ Generate a 2x3 plot layout with rows for affinity and expression. Args: dms_df (pd.DataFrame): Dataframe containing the data. dependent_var (str): Name of the dependent variable column. output_dir (str): Output directory for saving the plot. """ dms_df = dms_df.copy() dms_df.rename(columns={"delta_bind_CGG": "delta_affinity"}, inplace=True) dms_df.rename(columns={"delta_expr": "delta_expression"}, inplace=True) if timepoint == "": dependent_var = "mutation abundance" else: dependent_var = f"mutation abundance ({timepoint})" query_str="(single_nt == True) & (WT == False)" plt.figure(figsize=(13, 7)) axs = [plt.subplot(231), plt.subplot(232), plt.subplot(233)] plot_one_measured_var( axs, dms_df, query_str, dependent_var, "affinity", cmap=pc.affinity_dms.cmap, norm=pc.affinity_dms.norm ) axs = [plt.subplot(234), plt.subplot(235), plt.subplot(236)] plot_one_measured_var( axs, dms_df, query_str, dependent_var, "expression", cmap=pc.expression_dms.cmap, norm=pc.expression_dms.norm ) if timepoint != "": plt.suptitle(f"{timepoint}") plt.tight_layout() plt.savefig(f"{output_dir}/eventsVsrate{timepoint}.pdf") plt.show()

In [31]:

mutations_plot(dms_df)
mutations_plot(dms_df, timepoint="15-day")
mutations_plot(dms_df, timepoint="20-day")

mutations_plot(dms_df) mutations_plot(dms_df, timepoint="15-day") mutations_plot(dms_df, timepoint="20-day")

Spearman's ρ for log(mutation rate) vs mutation abundance: 0.6615
Spearman's ρ for Δaffinity vs mutation abundance: 0.2214

Spearman's ρ for log(mutation rate) vs mutation abundance: 0.6615
Spearman's ρ for Δexpression vs mutation abundance: 0.3967

Spearman's ρ for log(mutation rate) vs mutation abundance (15-day): 0.6507
Spearman's ρ for Δaffinity vs mutation abundance (15-day): 0.1946

Spearman's ρ for log(mutation rate) vs mutation abundance (15-day): 0.6507
Spearman's ρ for Δexpression vs mutation abundance (15-day): 0.3434

Spearman's ρ for log(mutation rate) vs mutation abundance (20-day): 0.6324
Spearman's ρ for Δaffinity vs mutation abundance (20-day): 0.2046

Spearman's ρ for log(mutation rate) vs mutation abundance (20-day): 0.6324
Spearman's ρ for Δexpression vs mutation abundance (20-day): 0.3821

In [32]:

query_str="(single_nt == True) & (WT == False)"
data = dms_df.query(query_str).dropna()

def minefield_scatter(ax, data, color_column):
    scatter = ax.scatter(
        data["delta_bind_CGG"],
        data["delta_expr"],
        c=data[color_column],
        cmap="RdBu_r",
        vmin=-np.abs(data[color_column]).max(),
        vmax=np.abs(data[color_column]).max(),
        s=20,
        linewidth=0  # Remove the edge lines
    )

    ax.axhline(y=-0.3, color="lightgray", linestyle="--")
    ax.axvline(x=-0.9, color="lightgray", linestyle="--")
    ax.axvline(x=-0.3, color="lightgray", linestyle="--")
    ax.axvline(x=0.3, color="lightgray", linestyle="--")

    sns.despine()

    cbar = fig.colorbar(scatter, ax=ax)
    cbar.set_label(color_column)

    ax.set_xlabel(r"$\Delta$ bind")
    ax.set_ylabel(r"$\Delta$ expr")


fig, ax = plt.subplots(figsize=(8, 6))
minefield_scatter(ax, data, "mutation enrichment")
fig.savefig(f"{output_dir}/minefield_with_enrichment_colors.pdf", bbox_inches="tight", dpi=300)

fig, ax = plt.subplots(figsize=(8, 6))
minefield_scatter(ax, data, "mutation enrichment (LMP2A)")
fig.savefig(f"{output_dir}/minefield_with_enrichment_colors_lmp2a.pdf", bbox_inches="tight", dpi=300)

query_str="(single_nt == True) & (WT == False)" data = dms_df.query(query_str).dropna() def minefield_scatter(ax, data, color_column): scatter = ax.scatter( data["delta_bind_CGG"], data["delta_expr"], c=data[color_column], cmap="RdBu_r", vmin=-np.abs(data[color_column]).max(), vmax=np.abs(data[color_column]).max(), s=20, linewidth=0 # Remove the edge lines ) ax.axhline(y=-0.3, color="lightgray", linestyle="--") ax.axvline(x=-0.9, color="lightgray", linestyle="--") ax.axvline(x=-0.3, color="lightgray", linestyle="--") ax.axvline(x=0.3, color="lightgray", linestyle="--") sns.despine() cbar = fig.colorbar(scatter, ax=ax) cbar.set_label(color_column) ax.set_xlabel(r"$\Delta$ bind") ax.set_ylabel(r"$\Delta$ expr") fig, ax = plt.subplots(figsize=(8, 6)) minefield_scatter(ax, data, "mutation enrichment") fig.savefig(f"{output_dir}/minefield_with_enrichment_colors.pdf", bbox_inches="tight", dpi=300) fig, ax = plt.subplots(figsize=(8, 6)) minefield_scatter(ax, data, "mutation enrichment (LMP2A)") fig.savefig(f"{output_dir}/minefield_with_enrichment_colors_lmp2a.pdf", bbox_inches="tight", dpi=300)

In [33]:

data = dms_df.query(query_str).dropna()
data = data[(data["mutation abundance (15-day)"] >= 5) | (data["mutation abundance (20-day)"] >= 5)]

fig, ax = plt.subplots(figsize=(8, 6))
vlim = 1
ax.scatter(data["mutation enrichment (20-day)"], data["mutation enrichment (LMP2A)"], c=data["delta_expr"], cmap="RdBu_r", vmin=-vlim, vmax=vlim, s=20)
ax.set_xlabel("mutation enrichment (20-day)")
ax.set_ylabel("mutation enrichment (LMP2A)")
cbar = fig.colorbar(ax.collections[0], ax=ax)
cbar.set_label(r"$\Delta$ expr")

ax.set_aspect('equal', adjustable='box')
ax.plot([-1, 1], [-1, 1], color="k", linestyle="--")

data = dms_df.query(query_str).dropna() data = data[(data["mutation abundance (15-day)"] >= 5) | (data["mutation abundance (20-day)"] >= 5)] fig, ax = plt.subplots(figsize=(8, 6)) vlim = 1 ax.scatter(data["mutation enrichment (20-day)"], data["mutation enrichment (LMP2A)"], c=data["delta_expr"], cmap="RdBu_r", vmin=-vlim, vmax=vlim, s=20) ax.set_xlabel("mutation enrichment (20-day)") ax.set_ylabel("mutation enrichment (LMP2A)") cbar = fig.colorbar(ax.collections[0], ax=ax) cbar.set_label(r"$\Delta$ expr") ax.set_aspect('equal', adjustable='box') ax.plot([-1, 1], [-1, 1], color="k", linestyle="--")

Out[33]:

[<matplotlib.lines.Line2D at 0x1539deca3340>]

In [34]:

def do_piecewise_regression(measured_var, n_breakpoints, enrichment_column="mutation enrichment"):
    query_str="(single_nt == True) & (WT == False)"
    data = dms_df.query(query_str).sort_values(by=f"delta_{measured_var}", key=lambda val: np.abs(val)).dropna()
    delta_thing = data[f"delta_{measured_var}"].to_numpy()
    enrichment = data[enrichment_column].to_numpy()

    if n_breakpoints is None:
        ms = piecewise_regression.ModelSelection(delta_thing, enrichment, max_breakpoints=5)
        return ms
    #else:
    pw_fit = piecewise_regression.Fit(delta_thing, enrichment, n_breakpoints=n_breakpoints)

    pw_fit.plot_data(color="grey", alpha=0.5)
    pw_fit.plot_fit(color="red", linewidth=4)
    pw_fit.plot_breakpoints()
    pw_fit.plot_breakpoint_confidence_intervals()
    pw_fit.summary()
    plt.xlabel(f"$\Delta$ {measured_var}")
    plt.ylabel(enrichment_column)
    fig = plt.gcf()
    return pw_fit, fig

do_piecewise_regression("bind_CGG", None)
pw_fit, fig = do_piecewise_regression("bind_CGG", 1)
fig.savefig(f"{output_dir}/piecewise_regression.bind.pdf")

def do_piecewise_regression(measured_var, n_breakpoints, enrichment_column="mutation enrichment"): query_str="(single_nt == True) & (WT == False)" data = dms_df.query(query_str).sort_values(by=f"delta_{measured_var}", key=lambda val: np.abs(val)).dropna() delta_thing = data[f"delta_{measured_var}"].to_numpy() enrichment = data[enrichment_column].to_numpy() if n_breakpoints is None: ms = piecewise_regression.ModelSelection(delta_thing, enrichment, max_breakpoints=5) return ms #else: pw_fit = piecewise_regression.Fit(delta_thing, enrichment, n_breakpoints=n_breakpoints) pw_fit.plot_data(color="grey", alpha=0.5) pw_fit.plot_fit(color="red", linewidth=4) pw_fit.plot_breakpoints() pw_fit.plot_breakpoint_confidence_intervals() pw_fit.summary() plt.xlabel(f"$\Delta$ {measured_var}") plt.ylabel(enrichment_column) fig = plt.gcf() return pw_fit, fig do_piecewise_regression("bind_CGG", None) pw_fit, fig = do_piecewise_regression("bind_CGG", 1) fig.savefig(f"{output_dir}/piecewise_regression.bind.pdf")

Running fit with n_breakpoint = 0 . . 
Running fit with n_breakpoint = 1 . . 

Running fit with n_breakpoint = 2 . . 

Running fit with n_breakpoint = 3 . . 

Running fit with n_breakpoint = 4 . . 

Running fit with n_breakpoint = 5 . . 

                 Breakpoint Model Comparision Results                 
====================================================================================================
n_breakpoints            BIC    converged          RSS 
----------------------------------------------------------------------------------------------------
0                    -1601.2         True       359.13 
1                    -1762.1         True       313.01 
2                    -1750.8         True       312.26 
3                                   False              
4                                   False              
5                                   False              

Min BIC (Bayesian Information Criterion) suggests best model

                    Breakpoint Regression Results                     
====================================================================================================
No. Observations                     1275
No. Model Parameters                    4
Degrees of Freedom                   1271
Res. Sum of Squares               313.006
Total Sum of Squares              430.826
R Squared                        0.273476
Adjusted R Squared               0.271188
Converged:                           True
====================================================================================================
====================================================================================================
                    Estimate      Std Err            t        P>|t|       [0.025       0.975]
----------------------------------------------------------------------------------------------------
const               -1.03708       0.0743      -13.955      2.75e-41      -1.1829     -0.89129
alpha1            -0.0957312       0.0554      -1.7281        0.0842     -0.20441     0.012951
beta1                1.27477       0.0949       13.438             -       1.0887       1.4609
breakpoint1        -0.540443       0.0521            -             -      -0.6427     -0.43818
----------------------------------------------------------------------------------------------------
These alphas(gradients of segments) are estimatedfrom betas(change in gradient)
----------------------------------------------------------------------------------------------------
alpha2               1.17903        0.077       15.312      1.07e-48        1.028       1.3301
====================================================================================================
Davies test for existence of at least 1 breakpoint: p=2.74038e-40 (e.g. p<0.05 means reject null hypothesis of no breakpoints at 5% significance)

In [35]:

do_piecewise_regression("expr", None)
pw_fit, fig = do_piecewise_regression("expr", 1)
fig.savefig(f"{output_dir}/piecewise_regression.expr.pdf")

do_piecewise_regression("expr", None) pw_fit, fig = do_piecewise_regression("expr", 1) fig.savefig(f"{output_dir}/piecewise_regression.expr.pdf")

Running fit with n_breakpoint = 0 . . 
Running fit with n_breakpoint = 1 . . 

Running fit with n_breakpoint = 2 . . 

Running fit with n_breakpoint = 3 . . 

Running fit with n_breakpoint = 4 . . 

Running fit with n_breakpoint = 5 . . 

                 Breakpoint Model Comparision Results                 
====================================================================================================
n_breakpoints            BIC    converged          RSS 
----------------------------------------------------------------------------------------------------
0                    -1712.4         True       329.13 
1                    -1799.2         True       304.04 
2                    -1792.9         True       302.13 
3                                   False              
4                                   False              
5                                   False              

Min BIC (Bayesian Information Criterion) suggests best model

                    Breakpoint Regression Results                     
====================================================================================================
No. Observations                     1275
No. Model Parameters                    4
Degrees of Freedom                   1271
Res. Sum of Squares               304.041
Total Sum of Squares              430.826
R Squared                        0.294284
Adjusted R Squared               0.292061
Converged:                           True
====================================================================================================
====================================================================================================
                    Estimate      Std Err            t        P>|t|       [0.025       0.975]
----------------------------------------------------------------------------------------------------
const              -0.643355       0.0477      -13.482      8.09e-39     -0.73697     -0.54974
alpha1                0.2532       0.0391       6.4718      1.38e-10      0.17645      0.32995
beta1                1.73327        0.221       7.8321             -       1.2991       2.1674
breakpoint1        -0.254603       0.0367            -             -     -0.32665     -0.18255
----------------------------------------------------------------------------------------------------
These alphas(gradients of segments) are estimatedfrom betas(change in gradient)
----------------------------------------------------------------------------------------------------
alpha2               1.98647        0.218       9.1199      2.86e-19       1.5591       2.4138
====================================================================================================
Davies test for existence of at least 1 breakpoint: p=3.69869e-19 (e.g. p<0.05 means reject null hypothesis of no breakpoints at 5% significance)

In [36]:

predicted_values = pw_fit.predict(data['delta_bind_CGG'].values)
data['enrichment affinity residual'] = data['mutation enrichment'] - predicted_values

predicted_values = pw_fit.predict(data['delta_bind_CGG'].values) data['enrichment affinity residual'] = data['mutation enrichment'] - predicted_values

In [37]:

data["delta_avidity"] = data["delta_bind_CGG"] + data["delta_expr"]

rho, _ = spearmanr(data["delta_bind_CGG"], data["mutation enrichment"])
print(f"Spearman's ρ for Δaffinity vs mutation enrichment: {rho:.4f}")

rho, _ = spearmanr(data["delta_expr"], data["mutation enrichment"])
print(f"Spearman's ρ for Δexpression vs mutation enrichment: {rho:.4f}")

rho, _ = spearmanr(data["delta_avidity"], data["mutation enrichment"])
print(f"Spearman's ρ for Δavidity vs mutation enrichment: {rho:.4f}")

rho, _ = spearmanr(data["delta_expr"], data["enrichment affinity residual"])
print(f"Spearman's ρ for Δexpression vs enrichment affinity residual: {rho:.4f}")

data["delta_avidity"] = data["delta_bind_CGG"] + data["delta_expr"] rho, _ = spearmanr(data["delta_bind_CGG"], data["mutation enrichment"]) print(f"Spearman's ρ for Δaffinity vs mutation enrichment: {rho:.4f}") rho, _ = spearmanr(data["delta_expr"], data["mutation enrichment"]) print(f"Spearman's ρ for Δexpression vs mutation enrichment: {rho:.4f}") rho, _ = spearmanr(data["delta_avidity"], data["mutation enrichment"]) print(f"Spearman's ρ for Δavidity vs mutation enrichment: {rho:.4f}") rho, _ = spearmanr(data["delta_expr"], data["enrichment affinity residual"]) print(f"Spearman's ρ for Δexpression vs enrichment affinity residual: {rho:.4f}")

Spearman's ρ for Δaffinity vs mutation enrichment: 0.4521
Spearman's ρ for Δexpression vs mutation enrichment: 0.3522
Spearman's ρ for Δavidity vs mutation enrichment: 0.5122
Spearman's ρ for Δexpression vs enrichment affinity residual: 0.0777

In [38]:

do_piecewise_regression("bind_CGG", None, "mutation enrichment (LMP2A)")
do_piecewise_regression("bind_CGG", 1, "mutation enrichment (LMP2A)")

do_piecewise_regression("bind_CGG", None, "mutation enrichment (LMP2A)") do_piecewise_regression("bind_CGG", 1, "mutation enrichment (LMP2A)")

Running fit with n_breakpoint = 0 . . 
Running fit with n_breakpoint = 1 . . 

Running fit with n_breakpoint = 2 . . 

Running fit with n_breakpoint = 3 . . 

Running fit with n_breakpoint = 4 . . 

Running fit with n_breakpoint = 5 . . 

                 Breakpoint Model Comparision Results                 
====================================================================================================
n_breakpoints            BIC    converged          RSS 
----------------------------------------------------------------------------------------------------
0                    -2198.7         True       224.76 
1                    -2289.9         True       206.91 
2                    -2277.9         True       206.53 
3                                   False              
4                                   False              
5                                   False              

Min BIC (Bayesian Information Criterion) suggests best model

                    Breakpoint Regression Results                     
====================================================================================================
No. Observations                     1275
No. Model Parameters                    4
Degrees of Freedom                   1271
Res. Sum of Squares                206.91
Total Sum of Squares              254.885
R Squared                        0.188225
Adjusted R Squared               0.185668
Converged:                           True
====================================================================================================
====================================================================================================
                    Estimate      Std Err            t        P>|t|       [0.025       0.975]
----------------------------------------------------------------------------------------------------
const              -0.454098       0.0481       -9.445      1.64e-20     -0.54842     -0.35978
alpha1            0.00515482       0.0396      0.13024         0.896    -0.072491     0.082801
beta1               0.846379       0.0853       9.9239             -      0.67906       1.0137
breakpoint1        -0.389044       0.0581            -             -     -0.50302     -0.27507
----------------------------------------------------------------------------------------------------
These alphas(gradients of segments) are estimatedfrom betas(change in gradient)
----------------------------------------------------------------------------------------------------
alpha2              0.851534       0.0755       11.272      3.74e-28      0.70332      0.99975
====================================================================================================
Davies test for existence of at least 1 breakpoint: p=6.44994e-23 (e.g. p<0.05 means reject null hypothesis of no breakpoints at 5% significance)

Out[38]:

(<piecewise_regression.main.Fit at 0x1539de90e440>,
 <Figure size 640x480 with 1 Axes>)

In [39]:

do_piecewise_regression("expr", None, "mutation enrichment (LMP2A)")
do_piecewise_regression("expr", 1, "mutation enrichment (LMP2A)")

do_piecewise_regression("expr", None, "mutation enrichment (LMP2A)") do_piecewise_regression("expr", 1, "mutation enrichment (LMP2A)")

Running fit with n_breakpoint = 0 . . 
Running fit with n_breakpoint = 1 . . 

Running fit with n_breakpoint = 2 . . 

Running fit with n_breakpoint = 3 . . 

Running fit with n_breakpoint = 4 . . 

Running fit with n_breakpoint = 5 . . 

                 Breakpoint Model Comparision Results                 
====================================================================================================
n_breakpoints            BIC    converged          RSS 
----------------------------------------------------------------------------------------------------
0                    -2158.5         True       231.95 
1                    -2203.5         True       221.41 
2                    -2192.6         True       220.83 
3                                   False              
4                                   False              
5                                   False              

Min BIC (Bayesian Information Criterion) suggests best model

                    Breakpoint Regression Results                     
====================================================================================================
No. Observations                     1275
No. Model Parameters                    4
Degrees of Freedom                   1271
Res. Sum of Squares               221.406
Total Sum of Squares              254.885
R Squared                        0.131352
Adjusted R Squared               0.128616
Converged:                           True
====================================================================================================
====================================================================================================
                    Estimate      Std Err            t        P>|t|       [0.025       0.975]
----------------------------------------------------------------------------------------------------
const               -0.30248       0.0369       -8.198      5.92e-16     -0.37486     -0.23009
alpha1             0.0961897       0.0316       3.0459       0.00237     0.034234      0.15815
beta1                1.28257        0.214       6.0055             -      0.86359       1.7015
breakpoint1        -0.200374       0.0388            -             -     -0.27652     -0.12423
----------------------------------------------------------------------------------------------------
These alphas(gradients of segments) are estimatedfrom betas(change in gradient)
----------------------------------------------------------------------------------------------------
alpha2               1.37876        0.211       6.5277      9.63e-11      0.96438       1.7931
====================================================================================================
Davies test for existence of at least 1 breakpoint: p=2.83692e-10 (e.g. p<0.05 means reject null hypothesis of no breakpoints at 5% significance)

Out[39]:

(<piecewise_regression.main.Fit at 0x1539dfcd8520>,
 <Figure size 640x480 with 1 Axes>)

In [40]:

do_piecewise_regression("expr", 1, "mutation enrichment (20-day)")

do_piecewise_regression("expr", 1, "mutation enrichment (20-day)")

                    Breakpoint Regression Results                     
====================================================================================================
No. Observations                     1275
No. Model Parameters                    4
Degrees of Freedom                   1271
Res. Sum of Squares               303.406
Total Sum of Squares              405.831
R Squared                        0.252382
Adjusted R Squared               0.250027
Converged:                           True
====================================================================================================
====================================================================================================
                    Estimate      Std Err            t        P>|t|       [0.025       0.975]
----------------------------------------------------------------------------------------------------
const              -0.717414       0.0631      -11.377      1.25e-28     -0.84112      -0.5937
alpha1              0.162411       0.0464       3.4996      0.000482     0.071365      0.25346
beta1                1.42969        0.165       8.6446             -       1.1052       1.7541
breakpoint1        -0.341803       0.0482            -             -     -0.43633     -0.24727
----------------------------------------------------------------------------------------------------
These alphas(gradients of segments) are estimatedfrom betas(change in gradient)
----------------------------------------------------------------------------------------------------
alpha2                1.5921        0.159        10.03      7.78e-23       1.2807       1.9035
====================================================================================================
Davies test for existence of at least 1 breakpoint: p=2.02555e-17 (e.g. p<0.05 means reject null hypothesis of no breakpoints at 5% significance)

Out[40]:

(<piecewise_regression.main.Fit at 0x1539dfe98940>,
 <Figure size 640x480 with 1 Axes>)

In [41]:

def plot_enrichment_vs_delta(measured_var, outcome_var, ax):
    regplot_lowess_ci(data=data,
            x=f"delta_{measured_var}", y=outcome_var ,
            ci_level=95, n_boot=100,
            scatter_kwargs=dict(s=13, alpha=0.5, color="grey"),
            line_kwargs=dict(color="xkcd:orange"),
            ax=ax,
            )
    ax.axhline(0, color="k", linestyle="--", linewidth=1)
    ax.axvline(0, color="k", linestyle="--", linewidth=1)
    ax.set_xlabel(fr"$\Delta$ {measured_var}")
    ax.set_ylabel(outcome_var)

fig, axs = plt.subplots(1, 3, figsize=(18, 6))
plot_enrichment_vs_delta("expr", "mutation enrichment", axs[0])
plot_enrichment_vs_delta("expr", "enrichment affinity residual", axs[1])
plot_enrichment_vs_delta("avidity", "mutation enrichment", axs[2])
plt.tight_layout()
plt.savefig(f"{output_dir}/three-lowess-supp.pdf")

def plot_enrichment_vs_delta(measured_var, outcome_var, ax): regplot_lowess_ci(data=data, x=f"delta_{measured_var}", y=outcome_var , ci_level=95, n_boot=100, scatter_kwargs=dict(s=13, alpha=0.5, color="grey"), line_kwargs=dict(color="xkcd:orange"), ax=ax, ) ax.axhline(0, color="k", linestyle="--", linewidth=1) ax.axvline(0, color="k", linestyle="--", linewidth=1) ax.set_xlabel(fr"$\Delta$ {measured_var}") ax.set_ylabel(outcome_var) fig, axs = plt.subplots(1, 3, figsize=(18, 6)) plot_enrichment_vs_delta("expr", "mutation enrichment", axs[0]) plot_enrichment_vs_delta("expr", "enrichment affinity residual", axs[1]) plot_enrichment_vs_delta("avidity", "mutation enrichment", axs[2]) plt.tight_layout() plt.savefig(f"{output_dir}/three-lowess-supp.pdf")

In [42]:

fig = make_dms_heatmap(dms_df, 'mutation enrichment', r"mutation enrichment", site_labels_pretty, cells_to_border)
plt.savefig(f"{output_dir}/heatmap.mut_enrichment.pdf")
plt.show()

fig = make_dms_heatmap(dms_df, 'mutation enrichment', r"mutation enrichment", site_labels_pretty, cells_to_border) plt.savefig(f"{output_dir}/heatmap.mut_enrichment.pdf") plt.show()

Event / rate Vs affinity (getting at selection sensitivity)

In [43]:

gain_thresh = 0.3
loss_thresh = -1
dms_df.loc[~dms_df.delta_bind_CGG.isna(), "affinity change stratum"] = "affinity neutral"
dms_df.loc[dms_df.delta_bind_CGG > gain_thresh, "affinity change stratum"] = "affinity gain"
dms_df.loc[dms_df.delta_bind_CGG < loss_thresh, "affinity change stratum"] = "affinity loss"

gain_thresh = 0.3 loss_thresh = -1 dms_df.loc[~dms_df.delta_bind_CGG.isna(), "affinity change stratum"] = "affinity neutral" dms_df.loc[dms_df.delta_bind_CGG > gain_thresh, "affinity change stratum"] = "affinity gain" dms_df.loc[dms_df.delta_bind_CGG < loss_thresh, "affinity change stratum"] = "affinity loss"

In [44]:

sns.scatterplot(data=dms_df, hue="affinity change stratum", y="mutation events", x="mutation rate", alpha=0.5, clip_on=False)
plt.xscale("symlog")
plt.yscale("symlog")
plt.xlim(0, None)
plt.ylim(0, None)
plt.show()

sns.scatterplot(data=dms_df, hue="affinity change stratum", y="mutation events", x="mutation rate", alpha=0.5, clip_on=False) plt.xscale("symlog") plt.yscale("symlog") plt.xlim(0, None) plt.ylim(0, None) plt.show()

In [45]:

affinity_incidence_rate_df = dms_df[["delta_bind_CGG", "delta_expr", "mutation events (20-day)", "mutation rate"]].copy()
affinity_incidence_rate_df["incidence / rate"] = affinity_incidence_rate_df["mutation events (20-day)"] / affinity_incidence_rate_df["mutation rate"]

bins = np.linspace(-1, 1, 9)
affinity_incidence_rate_df["affinity stratrum"] = pd.cut(affinity_incidence_rate_df["delta_bind_CGG"], bins=bins)
affinity_incidence_rate_df["expression stratrum"] = pd.cut(affinity_incidence_rate_df["delta_expr"], bins=bins)

plt.scatter(affinity_incidence_rate_df.delta_bind_CGG, affinity_incidence_rate_df.delta_expr, c=affinity_incidence_rate_df["incidence / rate"], alpha=0.5, s=10)
plt.show()

sns.scatterplot(data=affinity_incidence_rate_df, x="delta_bind_CGG", y="incidence / rate", alpha=0.5)
plt.yscale("log")
plt.show()
sns.scatterplot(data=affinity_incidence_rate_df, x="delta_expr", y="incidence / rate", alpha=0.5)
plt.show()

plt.figure(figsize=(10, 4))
sns.boxplot(data=affinity_incidence_rate_df, x="affinity stratrum", y="incidence / rate")
plt.show()
plt.figure(figsize=(10, 4))
sns.boxplot(data=affinity_incidence_rate_df, x="expression stratrum", y="incidence / rate")
plt.show()

sns.histplot(x=np.log10(affinity_incidence_rate_df["incidence / rate"]))
plt.show()

affinity_incidence_rate_df = dms_df[["delta_bind_CGG", "delta_expr", "mutation events (20-day)", "mutation rate"]].copy() affinity_incidence_rate_df["incidence / rate"] = affinity_incidence_rate_df["mutation events (20-day)"] / affinity_incidence_rate_df["mutation rate"] bins = np.linspace(-1, 1, 9) affinity_incidence_rate_df["affinity stratrum"] = pd.cut(affinity_incidence_rate_df["delta_bind_CGG"], bins=bins) affinity_incidence_rate_df["expression stratrum"] = pd.cut(affinity_incidence_rate_df["delta_expr"], bins=bins) plt.scatter(affinity_incidence_rate_df.delta_bind_CGG, affinity_incidence_rate_df.delta_expr, c=affinity_incidence_rate_df["incidence / rate"], alpha=0.5, s=10) plt.show() sns.scatterplot(data=affinity_incidence_rate_df, x="delta_bind_CGG", y="incidence / rate", alpha=0.5) plt.yscale("log") plt.show() sns.scatterplot(data=affinity_incidence_rate_df, x="delta_expr", y="incidence / rate", alpha=0.5) plt.show() plt.figure(figsize=(10, 4)) sns.boxplot(data=affinity_incidence_rate_df, x="affinity stratrum", y="incidence / rate") plt.show() plt.figure(figsize=(10, 4)) sns.boxplot(data=affinity_incidence_rate_df, x="expression stratrum", y="incidence / rate") plt.show() sns.histplot(x=np.log10(affinity_incidence_rate_df["incidence / rate"])) plt.show()

/opt/conda/envs/replay/lib/python3.10/site-packages/pandas/core/arraylike.py:399: RuntimeWarning: divide by zero encountered in log10
  result = getattr(ufunc, method)(*inputs, **kwargs)

In [46]:

affinity_incidence_rate_df

affinity_incidence_rate_df

Out[46]:

	delta_bind_CGG	delta_expr	mutation events (20-day)	mutation rate	incidence / rate	affinity stratrum	expression stratrum
mutation
E1(H)A	0.11933	-0.00246	4	0.000006	709451.963964	(0.0, 0.25]	(-0.25, 0.0]
E1(H)C	-0.01492	-0.12216	0	NaN	NaN	(-0.25, 0.0]	(-0.25, 0.0]
E1(H)D	0.01249	0.04874	7	0.000725	9660.529997	(0.0, 0.25]	(0.0, 0.25]
E1(H)E	0.00000	0.00000	0	NaN	NaN	(-0.25, 0.0]	(-0.25, 0.0]
E1(H)F	0.13433	-0.11379	0	NaN	NaN	(0.0, 0.25]	(-0.25, 0.0]
...	...	...	...	...	...	...	...
R128(L)S	0.01095	0.07344	0	NaN	NaN	(0.0, 0.25]	(0.0, 0.25]
R128(L)T	0.02081	0.08647	0	NaN	NaN	(0.0, 0.25]	(0.0, 0.25]
R128(L)V	-0.03418	0.04073	0	NaN	NaN	(-0.25, 0.0]	(0.0, 0.25]
R128(L)W	0.04423	-0.09192	0	NaN	NaN	(0.0, 0.25]	(-0.25, 0.0]
R128(L)Y	0.00737	0.01079	0	NaN	NaN	(0.0, 0.25]	(0.0, 0.25]

4400 rows × 7 columns

In [47]:

plt.figure(figsize=(10, 4))
sns.boxplot(x=affinity_incidence_rate_df["affinity stratrum"], y=np.log10(affinity_incidence_rate_df["incidence / rate"]))
plt.show()
plt.figure(figsize=(10, 4))
sns.boxplot(data=affinity_incidence_rate_df, x="expression stratrum", y="incidence / rate")
plt.show()

plt.figure(figsize=(10, 4)) sns.boxplot(x=affinity_incidence_rate_df["affinity stratrum"], y=np.log10(affinity_incidence_rate_df["incidence / rate"])) plt.show() plt.figure(figsize=(10, 4)) sns.boxplot(data=affinity_incidence_rate_df, x="expression stratrum", y="incidence / rate") plt.show()

/opt/conda/envs/replay/lib/python3.10/site-packages/pandas/core/arraylike.py:399: RuntimeWarning: divide by zero encountered in log10
  result = getattr(ufunc, method)(*inputs, **kwargs)

In [48]:

plt.figure(figsize=(3, 4))

ax = plt.subplot(211)
sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", bins=40, stat="probability", element="step", fill=False, label="uniform")
sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", bins=40, weights=dms_df.loc[dms_df.single_nt, "mutation rate"], stat="probability", element="step", fill=False, label="biased")
# plt.xlim(-2, 2)
plt.xlabel(r"$\Delta$ affinity")
# plt.yscale("log")
plt.legend(title="mutation process", fontsize=10)

plt.subplot(212)
sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr", bins=40, stat="probability", element="step", fill=False, label="uniform")
sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr", bins=40, weights=dms_df.loc[dms_df.single_nt, "mutation rate"], stat="probability", element="step", fill=False, label="biased")
plt.xlim(-2, 2)
plt.xlabel(r"$\Delta$ expression")
# plt.yscale("log")


# sns.jointplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", y="delta_expr")


plt.tight_layout()
plt.savefig(f"{output_dir}/mutation-bias.pdf")
plt.show()

plt.figure(figsize=(3, 4)) ax = plt.subplot(211) sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", bins=40, stat="probability", element="step", fill=False, label="uniform") sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", bins=40, weights=dms_df.loc[dms_df.single_nt, "mutation rate"], stat="probability", element="step", fill=False, label="biased") # plt.xlim(-2, 2) plt.xlabel(r"$\Delta$ affinity") # plt.yscale("log") plt.legend(title="mutation process", fontsize=10) plt.subplot(212) sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr", bins=40, stat="probability", element="step", fill=False, label="uniform") sns.histplot(data=dms_df.loc[dms_df.single_nt], x="delta_expr", bins=40, weights=dms_df.loc[dms_df.single_nt, "mutation rate"], stat="probability", element="step", fill=False, label="biased") plt.xlim(-2, 2) plt.xlabel(r"$\Delta$ expression") # plt.yscale("log") # sns.jointplot(data=dms_df.loc[dms_df.single_nt], x="delta_bind_CGG", y="delta_expr") plt.tight_layout() plt.savefig(f"{output_dir}/mutation-bias.pdf") plt.show()

In [49]:

plt.figure(figsize=(30, 12))
plt.subplot(411)
sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="delta_bind_CGG",
            hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1],
            order=sorted(dms_df.site.unique()), dodge=False)
plt.axhline(0, c="k")
plt.ylabel(r"$\Delta$ affinity")
plt.xticks(rotation=90)

plt.subplot(412)
sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="delta_expr",
            hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1],
            order=sorted(dms_df.site.unique()), dodge=False)
plt.axhline(0, c="k")
plt.ylabel(r"$\Delta$ expression")
plt.xticks(rotation=90)

plt.subplot(413)
g = sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="mutation rate",
            hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1],
            order=sorted(dms_df.site.unique()), dodge=False)
plt.xticks(rotation=90)
g.legend_.remove()

plt.subplot(414)
g = sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="mutation events",
            hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1],
            order=sorted(dms_df.site.unique()), dodge=False)
g.legend_.remove()
plt.xticks(rotation=90)

plt.tight_layout()
plt.savefig(f"{output_dir}/regions.pdf")
plt.show()

plt.figure(figsize=(30, 12)) plt.subplot(411) sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="delta_bind_CGG", hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1], order=sorted(dms_df.site.unique()), dodge=False) plt.axhline(0, c="k") plt.ylabel(r"$\Delta$ affinity") plt.xticks(rotation=90) plt.subplot(412) sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="delta_expr", hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1], order=sorted(dms_df.site.unique()), dodge=False) plt.axhline(0, c="k") plt.ylabel(r"$\Delta$ expression") plt.xticks(rotation=90) plt.subplot(413) g = sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="mutation rate", hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1], order=sorted(dms_df.site.unique()), dodge=False) plt.xticks(rotation=90) g.legend_.remove() plt.subplot(414) g = sns.barplot(data=dms_df.loc[dms_df.single_nt], x="site", y="mutation events", hue=dms_df.loc[dms_df.single_nt].annotation.str[:-1], order=sorted(dms_df.site.unique()), dodge=False) g.legend_.remove() plt.xticks(rotation=90) plt.tight_layout() plt.savefig(f"{output_dir}/regions.pdf") plt.show()

Binding Vs Expression Vs structure¶

In [50]:

# copypasta from polyclonal
def extract_atom_locations(
    input_pdbfile,
    target_chains,
    target_atom="CA",
):
    # read PDB, catch warnings about discontinuous chains
    with warnings.catch_warnings():
        warnings.simplefilter(
            "ignore", category=Bio.PDB.PDBExceptions.PDBConstructionWarning
        )
        pdb = Bio.PDB.PDBParser().get_structure("_", input_pdbfile)

    # get the chains out of the PDB
    chains = list(pdb.get_chains())
    chain_ids = [chain.id for chain in chains]

    # make sure the target chains are in the PDB
    for chain in target_chains:
        if chain not in chain_ids:
            raise ValueError(f"{chain} not in {input_pdbfile}")

    # make a list of chains to extract atom locations from
    chains_to_use = []
    for i, chain in enumerate(chain_ids):
        if chain in target_chains:
            chains_to_use.append(chains[i])

    # extract atom locations from target chains
    chain_list = []
    residue_list = []
    x_list = []
    y_list = []
    z_list = []
    for chain in chains_to_use:
        for residue in chain.get_residues():
            residue_number = residue.get_id()[1]
            atoms = residue.get_atoms()
            for atom in atoms:
                if atom.get_id() == target_atom:
                    x, y, z = atom.get_coord()
                    x_list.append(x)
                    y_list.append(y)
                    z_list.append(z)
                    residue_list.append(residue_number)
                    chain_list.append(chain.id)

    # write output
    output = pd.DataFrame(
        {
            "chain": chain_list,
            "site": residue_list,
            "x": x_list,
            "y": y_list,
            "z": z_list,
        }
    )

    return output.reset_index(drop=True)

# copypasta from polyclonal def extract_atom_locations( input_pdbfile, target_chains, target_atom="CA", ): # read PDB, catch warnings about discontinuous chains with warnings.catch_warnings(): warnings.simplefilter( "ignore", category=Bio.PDB.PDBExceptions.PDBConstructionWarning ) pdb = Bio.PDB.PDBParser().get_structure("_", input_pdbfile) # get the chains out of the PDB chains = list(pdb.get_chains()) chain_ids = [chain.id for chain in chains] # make sure the target chains are in the PDB for chain in target_chains: if chain not in chain_ids: raise ValueError(f"{chain} not in {input_pdbfile}") # make a list of chains to extract atom locations from chains_to_use = [] for i, chain in enumerate(chain_ids): if chain in target_chains: chains_to_use.append(chains[i]) # extract atom locations from target chains chain_list = [] residue_list = [] x_list = [] y_list = [] z_list = [] for chain in chains_to_use: for residue in chain.get_residues(): residue_number = residue.get_id()[1] atoms = residue.get_atoms() for atom in atoms: if atom.get_id() == target_atom: x, y, z = atom.get_coord() x_list.append(x) y_list.append(y) z_list.append(z) residue_list.append(residue_number) chain_list.append(chain.id) # write output output = pd.DataFrame( { "chain": chain_list, "site": residue_list, "x": x_list, "y": y_list, "z": z_list, } ) return output.reset_index(drop=True)

In [51]:

# def residue_depth(
#     input_pdbfile,
#     target_chain,
# ):
#     parser = Bio.PDB.PDBParser()
#     # read PDB, catch warnings about discontinuous chains
#     with warnings.catch_warnings():
#         warnings.simplefilter(
#             "ignore", category=Bio.PDB.PDBExceptions.PDBConstructionWarning
#         )
#         pdb = Bio.PDB.PDBParser().get_structure("_", input_pdbfile)
#     model = pdb[0]
#     print(list(pdb.get_chains()))
#     rd = Bio.PDB.ResidueDepth(model)
# #     print(rd)
    
# residue_depth("combined_ch2_eh2-coot_IMGT.pdb", "H")

# def residue_depth( # input_pdbfile, # target_chain, # ): # parser = Bio.PDB.PDBParser() # # read PDB, catch warnings about discontinuous chains # with warnings.catch_warnings(): # warnings.simplefilter( # "ignore", category=Bio.PDB.PDBExceptions.PDBConstructionWarning # ) # pdb = Bio.PDB.PDBParser().get_structure("_", input_pdbfile) # model = pdb[0] # print(list(pdb.get_chains())) # rd = Bio.PDB.ResidueDepth(model) # # print(rd) # residue_depth("combined_ch2_eh2-coot_IMGT.pdb", "H")

In [52]:

coords_H = extract_atom_locations(open(pdb), "H").set_index("site").drop("chain", axis=1)
coords_L = extract_atom_locations(open(pdb), "L").set_index("site").drop("chain", axis=1)
coords_A = extract_atom_locations(open(pdb), "A").set_index("site").drop("chain", axis=1)
coords_B = extract_atom_locations(open(pdb), "B").set_index("site").drop("chain", axis=1)
coords_C = extract_atom_locations(open(pdb), "C").set_index("site").drop("chain", axis=1)
coords_D = extract_atom_locations(open(pdb), "D").set_index("site").drop("chain", axis=1)

coords_H = extract_atom_locations(open(pdb), "H").set_index("site").drop("chain", axis=1) coords_L = extract_atom_locations(open(pdb), "L").set_index("site").drop("chain", axis=1) coords_A = extract_atom_locations(open(pdb), "A").set_index("site").drop("chain", axis=1) coords_B = extract_atom_locations(open(pdb), "B").set_index("site").drop("chain", axis=1) coords_C = extract_atom_locations(open(pdb), "C").set_index("site").drop("chain", axis=1) coords_D = extract_atom_locations(open(pdb), "D").set_index("site").drop("chain", axis=1)

Add distance info to dms_df

In [53]:

coords_L

coords_L

Out[53]:

	x	y	z
site
1	137.317993	157.432999	169.117004
2	135.832993	154.018005	169.938995
3	134.294006	151.919998	172.697006
4	135.925995	148.505005	172.539001
5	134.113007	146.154007	174.936996
...	...	...	...
119	139.772995	135.639999	181.841003
120	140.578003	131.979996	180.955002
121	139.048996	129.811996	183.735001
122	139.682007	126.005997	184.268005
123	136.938995	123.848000	185.878998

107 rows × 3 columns

In [54]:

for coords_Ab, chain in zip((coords_H, coords_L), "HL"):
    for i in coords_Ab.index:
        min_dist = np.inf
        for coords_Ag in (coords_A, coords_B, coords_C, coords_D):
            chain_min = np.linalg.norm(coords_Ab.loc[i] - coords_Ag, axis=1).min()
            if chain_min < min_dist:
                min_dist = chain_min
        dms_df.loc[(dms_df.chain == chain) & (dms_df.position_IMGT == i), "distance to antigen"] = min_dist

dms_df

for coords_Ab, chain in zip((coords_H, coords_L), "HL"): for i in coords_Ab.index: min_dist = np.inf for coords_Ag in (coords_A, coords_B, coords_C, coords_D): chain_min = np.linalg.norm(coords_Ab.loc[i] - coords_Ag, axis=1).min() if chain_min < min_dist: min_dist = chain_min dms_df.loc[(dms_df.chain == chain) & (dms_df.position_IMGT == i), "distance to antigen"] = min_dist dms_df

Out[54]:

	target	wildtype	position	position_IMGT	chain	annotation	mutant	codon	single_nt	bind_CGG	...	mutation abundance (LMP2A)	mutation events	mutation abundance	mutation rate	mutation enrichment	mutation enrichment (15-day)	mutation enrichment (20-day)	mutation enrichment (LMP2A)	affinity change stratum	distance to antigen
mutation
E1(H)A	CGG_naive	E	1	1	H	FWRH1	A	GAG	True	8.74239	...	0	6	6	0.000006	1.003672	0.653329	0.883009	-0.010224	affinity neutral	9.796122
E1(H)C	CGG_naive	E	1	1	H	FWRH1	C	GAG	False	8.60813	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	9.796122
E1(H)D	CGG_naive	E	1	1	H	FWRH1	D	GAG	True	8.63554	...	4	10	57	0.000725	0.479223	-0.337929	0.658904	0.345565	affinity neutral	9.796122
E1(H)E	CGG_naive	E	1	1	H	FWRH1	E	GAG	True	8.62305	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	9.796122
E1(H)F	CGG_naive	E	1	1	H	FWRH1	F	GAG	False	8.75738	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	9.796122
...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...	...
R128(L)S	CGG_naive	R	235	128	L	FWRL4	S	CGT	True	8.63400	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	NaN
R128(L)T	CGG_naive	R	235	128	L	FWRL4	T	CGT	False	8.64386	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	NaN
R128(L)V	CGG_naive	R	235	128	L	FWRL4	V	CGT	False	8.58887	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	NaN
R128(L)W	CGG_naive	R	235	128	L	FWRL4	W	CGT	False	8.66728	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	NaN
R128(L)Y	CGG_naive	R	235	128	L	FWRL4	Y	CGT	False	8.63043	...	0	0	0	NaN	NaN	NaN	NaN	NaN	affinity neutral	NaN

4400 rows × 36 columns

In [55]:

dms_df.to_csv(f"{output_dir}/data.csv")

dms_df.to_csv(f"{output_dir}/data.csv")

In [56]:

plt.figure(figsize=(5, 4))
plt.scatter(dms_df.delta_expr, y=dms_df.delta_bind_CGG, c=dms_df["distance to antigen"],
            s=2, alpha=0.5,
#             cmap="viridis",
#             vmin=0,
            vmax=15
           )
plt.axvline(0, ls="--", c="k", lw=1)
plt.axhline(0, ls="--", c="k", lw=1)
plt.xlabel(r"$\Delta$ expression")
plt.ylabel(r"$\Delta$ affinity")
plt.colorbar(label=r"distance to antigen ($\AA$)")
# plt.savefig(f"{output_dir}/bind-expr-dist.pdf")
plt.show()

plt.figure(figsize=(5, 4)) plt.scatter(dms_df.delta_expr, y=dms_df.delta_bind_CGG, c=dms_df["distance to antigen"], s=2, alpha=0.5, # cmap="viridis", # vmin=0, vmax=15 ) plt.axvline(0, ls="--", c="k", lw=1) plt.axhline(0, ls="--", c="k", lw=1) plt.xlabel(r"$\Delta$ expression") plt.ylabel(r"$\Delta$ affinity") plt.colorbar(label=r"distance to antigen ($\AA$)") # plt.savefig(f"{output_dir}/bind-expr-dist.pdf") plt.show()

In [57]:

plt.figure(figsize=(4, 4))
sns.histplot(data=dms_df, x="delta_bind_CGG", y="distance to antigen", bins=50)
plt.axvline(0, ls="--", c="k", lw=1)
plt.xlabel(r"$\Delta$ affinity")
plt.ylabel(r"distance to antigen ($\AA$)")
plt.savefig(f"{output_dir}/bind-dist.pdf")
plt.show()

plt.figure(figsize=(4, 4)) sns.histplot(data=dms_df, x="delta_bind_CGG", y="distance to antigen", bins=50) plt.axvline(0, ls="--", c="k", lw=1) plt.xlabel(r"$\Delta$ affinity") plt.ylabel(r"distance to antigen ($\AA$)") plt.savefig(f"{output_dir}/bind-dist.pdf") plt.show()

In [58]:

plt.figure(figsize=(50, 4))
sns.heatmap(pd.DataFrame(dms_df.pivot(index="mutant", columns="site", values="distance to antigen").iloc[0, :]).T,
            vmax=15, cmap="viridis",
            cbar_kws=dict(label=r"distance to antigen ($\AA$)"), linewidths=.1, square=True)
plt.savefig(f"{output_dir}/bind-heatmap-distances-row.pdf")
plt.show()

plt.figure(figsize=(50, 4)) sns.heatmap(pd.DataFrame(dms_df.pivot(index="mutant", columns="site", values="distance to antigen").iloc[0, :]).T, vmax=15, cmap="viridis", cbar_kws=dict(label=r"distance to antigen ($\AA$)"), linewidths=.1, square=True) plt.savefig(f"{output_dir}/bind-heatmap-distances-row.pdf") plt.show()

High-incidence mutations¶

In [59]:

dms_df[["mutation events", "delta_bind_CGG"]].sort_values("mutation events", ascending=False)[:20]

dms_df[["mutation events", "delta_bind_CGG"]].sort_values("mutation events", ascending=False)[:20]

Out[59]:

	mutation events	delta_bind_CGG
mutation
S57(H)N	424	0.12761
S64(H)N	294	0.05093
N108(L)K	245	0.85965
S109(L)N	214	0.01231
Q105(L)H	214	0.38030
A105(H)G	195	0.44629
S109(L)R	183	0.79500
E101(L)D	181	-0.11838
Y42(L)F	172	-0.03927
S64(H)R	171	0.87785
S64(H)T	144	-0.02745
L124(L)V	142	0.08206
R67(L)K	136	0.15471
T96(H)S	134	0.04358
A40(L)G	132	0.80414
S92(L)N	126	0.03607
Y103(H)F	123	0.13457
G36(L)D	122	-0.59849
S109(L)T	119	0.01760
L50(H)F	111	-0.00532

In [60]:

# high_incidence = ["H-057", "H-064", "L-108", "L-109"]
high_incidence = dms_df.loc[dms_df["mutation events"] > dms_df["mutation events"].quantile(0.99)].site.unique()

vmin = -1
vmax = 1

plt.figure(figsize=(20, 4))
sns.heatmap(bind_df[high_incidence], center=0, vmin=vmin, vmax=vmax, annot=annot[high_incidence], fmt="", annot_kws=dict(fontweight="bold"),
            cmap="seismic_r", cbar_kws=dict(label=r"$-\Delta\log_{10} K_D$"), linewidths=.1, square=True)
plt.xlabel("site")
plt.ylabel("amino acid")
plt.tight_layout()
# plt.savefig(f"{output_dir}/high-incidence.pdf")
plt.show()

# high_incidence = ["H-057", "H-064", "L-108", "L-109"] high_incidence = dms_df.loc[dms_df["mutation events"] > dms_df["mutation events"].quantile(0.99)].site.unique() vmin = -1 vmax = 1 plt.figure(figsize=(20, 4)) sns.heatmap(bind_df[high_incidence], center=0, vmin=vmin, vmax=vmax, annot=annot[high_incidence], fmt="", annot_kws=dict(fontweight="bold"), cmap="seismic_r", cbar_kws=dict(label=r"$-\Delta\log_{10} K_D$"), linewidths=.1, square=True) plt.xlabel("site") plt.ylabel("amino acid") plt.tight_layout() # plt.savefig(f"{output_dir}/high-incidence.pdf") plt.show()

Pairwise epistasis¶

In [61]:

from scipy.stats import fisher_exact
from statsmodels.stats.multitest import fdrcorrection

from scipy.stats import fisher_exact from statsmodels.stats.multitest import fdrcorrection

In [62]:

mutations =  dms_df.index.unique()

n_mutations = 0
mutation_counts = pd.Series(0, index=mutations)

epi_df = pd.DataFrame(np.zeros((len(mutations), len(mutations))), index=mutations, columns=mutations)

for gc, tree in trees.items():
    print(gc, end="       \r")
    for node in tree.tree.iter_descendants(strategy="preorder"):
        parent_mutations = set(mutation for mutation in node.up.mutations)
        new_mutations = set(mutation for mutation in node.mutations) - parent_mutations
        if any(mutation.endswith("*") for mutation in parent_mutations.union(new_mutations)):
            continue
        for new_mutation in new_mutations:
            n_mutations += 1
            mutation_counts[new_mutation] += 1
        for background_mutation in parent_mutations:
            for new_mutation in new_mutations:
                epi_df.loc[background_mutation, new_mutation] += 1

volcano_dat = []
max_sig_to_print = 10
count = 0
for background_mutation in mutations:
    background_total = epi_df.loc[background_mutation, :].sum()
    if background_total >= 5:
        for new_mutation in mutations:
            if background_mutation[:-1] != new_mutation[:-1] and mutation_counts[new_mutation] >= 5:
                # print(background_mutation, end="       \r")
                table = np.zeros((2, 2), dtype=int)
                table[0, 1] = mutation_counts[new_mutation] - epi_df.loc[background_mutation, new_mutation]
                table[1, 0] = background_total - epi_df.loc[background_mutation, new_mutation]
                table[1, 1] = epi_df.loc[background_mutation, new_mutation]
                table[0, 0] = n_mutations - table.sum()
                assert table.sum() == n_mutations
                
                odds_ratio, p_value = fisher_exact(table, alternative="two-sided")
                if p_value < 1e-6:
                    count += 1
                    if count <= max_sig_to_print:
                        print(background_mutation, new_mutation, odds_ratio, p_value)
                        count += 1
                volcano_dat.append([background_mutation, new_mutation, table, odds_ratio, p_value])

volcano_df = pd.DataFrame(volcano_dat, columns=["background mutation", "mutation event", "contingency table", "odds ratio", "p-value"])

_, volcano_df["FDR"] = fdrcorrection(volcano_df["p-value"], method="p")

FDR_tresh = 0.05
volcano_df[f"FDR < {FDR_tresh}"] = volcano_df.FDR < FDR_tresh

epi_muts = volcano_df[volcano_df[f"FDR < {FDR_tresh}"]].sort_values("p-value")
epi_muts

mutations = dms_df.index.unique() n_mutations = 0 mutation_counts = pd.Series(0, index=mutations) epi_df = pd.DataFrame(np.zeros((len(mutations), len(mutations))), index=mutations, columns=mutations) for gc, tree in trees.items(): print(gc, end=" \r") for node in tree.tree.iter_descendants(strategy="preorder"): parent_mutations = set(mutation for mutation in node.up.mutations) new_mutations = set(mutation for mutation in node.mutations) - parent_mutations if any(mutation.endswith("*") for mutation in parent_mutations.union(new_mutations)): continue for new_mutation in new_mutations: n_mutations += 1 mutation_counts[new_mutation] += 1 for background_mutation in parent_mutations: for new_mutation in new_mutations: epi_df.loc[background_mutation, new_mutation] += 1 volcano_dat = [] max_sig_to_print = 10 count = 0 for background_mutation in mutations: background_total = epi_df.loc[background_mutation, :].sum() if background_total >= 5: for new_mutation in mutations: if background_mutation[:-1] != new_mutation[:-1] and mutation_counts[new_mutation] >= 5: # print(background_mutation, end=" \r") table = np.zeros((2, 2), dtype=int) table[0, 1] = mutation_counts[new_mutation] - epi_df.loc[background_mutation, new_mutation] table[1, 0] = background_total - epi_df.loc[background_mutation, new_mutation] table[1, 1] = epi_df.loc[background_mutation, new_mutation] table[0, 0] = n_mutations - table.sum() assert table.sum() == n_mutations odds_ratio, p_value = fisher_exact(table, alternative="two-sided") if p_value < 1e-6: count += 1 if count <= max_sig_to_print: print(background_mutation, new_mutation, odds_ratio, p_value) count += 1 volcano_dat.append([background_mutation, new_mutation, table, odds_ratio, p_value]) volcano_df = pd.DataFrame(volcano_dat, columns=["background mutation", "mutation event", "contingency table", "odds ratio", "p-value"]) _, volcano_df["FDR"] = fdrcorrection(volcano_df["p-value"], method="p") FDR_tresh = 0.05 volcano_df[f"FDR < {FDR_tresh}"] = volcano_df.FDR < FDR_tresh epi_muts = volcano_df[volcano_df[f"FDR < {FDR_tresh}"]].sort_values("p-value") epi_muts

D15_M1_GC1       
D15_M1_GC2       
D15_M1_GC3       
D15_M2_GC4       
D15_M3_GC5       
D15_M3_GC6       
D15_M4_GC7       
D15_M4_GC8       
D15_M5_GC9       

D15_M5_GC10       
D15_M6_GC11       
D15_M6_GC12       
D15_M6_GC13       

D15_M6_GC14       
D15_M7_GC15       
D15_M7_GC16       
D15_M8_GC17       
D15_M9_GC18       
D15_M9_GC19       

D15_M10_GC20       
D15_M10_GC21       
D15_M10_GC22       
D15_M10_GC23       
D15_M11_GC24       

D15_M11_GC25       
D15_M11_GC26       
D15_M12_GC27       
D15_M12_GC28       
D15_M12_GC29       

D15_M13_GC30       
D15_M13_GC31       
D15_M13_GC32       
D15_M13_GC33       
D15_M14_GC34       
D15_M14_GC35       
D15_M14_GC36       

D15_M14_GC37       
D15_M15_GC38       
D15_M16_GC39       
D15_M16_GC40       
D15_M17_GC41       
D15_M17_GC42       
D15_M17_GC43       
D15_M17_GC44       

D15_M17_GC45       
D15_M18_GC46       
D15_M18_GC47       
D15_M18_GC48       
D15_M19_GC49       
D15_M19_GC50       

D15_M19_GC51       
D15_M19_GC52       
D20_M20_GC53       
D20_M20_GC54       
D20_M20_GC55       
D20_M20_GC56       

D20_M20_GC57       
D20_M20_GC58       
D20_M20_GC59       

D20_M20_GC60       
D20_M20_GC61       
D20_M20_GC62       
D20_M20_GC63       

D20_M20_GC64       
D20_M20_GC65       
D20_M20_GC66       
D20_M20_GC67       
D20_M20_GC68       

D20_M20_GC69       
D20_M21_GC70       
D20_M21_GC71       
D20_M21_GC72       
D20_M21_GC73       

D20_M21_GC74       
D20_M21_GC75       
D20_M21_GC76       
D20_M21_GC77       

D20_M21_GC78       
D20_M21_GC79       
D20_M21_GC80       
D20_M21_GC81       
D20_M21_GC82       
D20_M21_GC83       

D20_M21_GC84       
D20_M22_GC85       
D20_M22_GC86       
D20_M22_GC87       
D20_M22_GC88       

D20_M22_GC89       
D20_M22_GC90       
D20_M22_GC91       
D20_M22_GC92       
D20_M22_GC93       

D20_M22_GC94       
D20_M22_GC95       
D20_M22_GC96       
D20_M22_GC97       

D20_M22_GC98       
D20_M22_GC99       
D20_M23_GC100       
D20_M23_GC101       
D20_M23_GC102       
D20_M23_GC103       

D20_M23_GC104       
D20_M23_GC105       
D20_M23_GC106       
D20_M23_GC107       
D20_M23_GC108       

D20_M23_GC109       
D20_M24_GC110       
D20_M24_GC111       
D20_M24_GC112       
D20_M24_GC113       

D20_M24_GC114       
D20_M24_GC115       
D20_M25_GC116       
D20_M25_GC117       

D20_M25_GC118       
D20_M25_GC119       
W10_M26_GC120       
W10_M26_GC121       
W10_M26_GC122       
W10_M26_GC123       

W10_M27_GC124       
W10_M27_GC125       

W10_M27_GC126       

W10_M27_GC127       

W10_M27_GC128       

W10_M27_GC129       
D20_M28_GC130       
D20_M28_GC132       
D20_M28_GC133       

D20_M28_GC134       
D20_M28_GC136       
D20_M28_GC137       
D20_M28_GC138       
D20_M28_GC139       
D20_M28_GC140       

D20_M28_GC141       
D20_M29_GC142       
D20_M29_GC143       
D20_M29_GC144       

D20_M29_GC145       
D20_M29_GC146       
D20_M29_GC147       
D20_M29_GC148       

D20_M29_GC149       

A105(H)G Y42(L)H 0.263140163014973 7.205297810321075e-07

N108(L)K A105(H)G 0.22044706653556212 1.2081848614716333e-09

Out[62]:

	background mutation	mutation event	contingency table	odds ratio	p-value	FDR	FDR < 0.05
278666	N108(L)K	A105(H)G	[[15286, 226], [3375, 11]]	0.220447	1.208185e-09	0.000368	True

In [63]:

plt.figure(figsize=(4, 4))
ax = sns.scatterplot(data=volcano_df, x="odds ratio", y="p-value", hue=f"FDR < {FDR_tresh}", palette=["lightgrey", "red"])
ax.invert_yaxis()
plt.axvline(1, ls="--", c="k", lw=1)
plt.axhline(1, ls="--", c="k", lw=1)
# plt.axhline(volcano_df.loc[volcano_df[f"FDR < {FDR_tresh}"], "p-value"].max(), ls="--", c="red", lw=1)
plt.xscale("log", base=2)
plt.yscale("log")
plt.tight_layout()
plt.savefig(f"{output_dir}/epistasis.volcano.pdf")
plt.show()

plt.figure(figsize=(4, 4)) ax = sns.scatterplot(data=volcano_df, x="odds ratio", y="p-value", hue=f"FDR < {FDR_tresh}", palette=["lightgrey", "red"]) ax.invert_yaxis() plt.axvline(1, ls="--", c="k", lw=1) plt.axhline(1, ls="--", c="k", lw=1) # plt.axhline(volcano_df.loc[volcano_df[f"FDR < {FDR_tresh}"], "p-value"].max(), ls="--", c="red", lw=1) plt.xscale("log", base=2) plt.yscale("log") plt.tight_layout() plt.savefig(f"{output_dir}/epistasis.volcano.pdf") plt.show()

In [64]:

epi_muts.to_csv(f"{output_dir}/epistatic-pairs.csv", index=False)

epi_muts.to_csv(f"{output_dir}/epistatic-pairs.csv", index=False)

Where are the above mutations in our scatters?

In [65]:

background_indicator = dms_df.index.isin(epi_muts["background mutation"])
enabled_indicator = dms_df.index.isin(epi_muts["mutation event"])
indicator = ["other"] * len(dms_df)
for i in range(len(indicator)):
    if background_indicator[i]:
        indicator[i] = "epistatic background mutation"
    elif enabled_indicator[i]:
        indicator[i] = "epistatically enabled mutation"
indicator = pd.Series(indicator, index=dms_df.index, name="mutation type")

# indicator = dms_df.index == "A40(L)G" #"A40(L)G"

background_indicator = dms_df.index.isin(epi_muts["background mutation"]) enabled_indicator = dms_df.index.isin(epi_muts["mutation event"]) indicator = ["other"] * len(dms_df) for i in range(len(indicator)): if background_indicator[i]: indicator[i] = "epistatic background mutation" elif enabled_indicator[i]: indicator[i] = "epistatically enabled mutation" indicator = pd.Series(indicator, index=dms_df.index, name="mutation type") # indicator = dms_df.index == "A40(L)G" #"A40(L)G"

In [66]:

plt.figure(figsize=(6, 3))

ax = sns.scatterplot(x=dms_df["mutation rate"], y=dms_df["mutation events"], hue=indicator, s=20, alpha=0.7,
                     hue_order=("epistatic background mutation", "epistatically enabled mutation", "other"),
                     palette=("C0", "C1", "lightgrey"),
                     clip_on=False, edgecolors="k", linewidths=0.1)
# plt.xlabel("mutation rate")
plt.ylabel("mutation events")
linthresh_x = 1e-5
linthresh_y = 1.0
plt.xscale("symlog", linthresh=linthresh_x, linscale=.5)
plt.yscale("symlog", linthresh=linthresh_y, linscale=.2)
plt.xlim(0, None)
plt.ylim(0, None)
sns.move_legend(ax, "upper left", bbox_to_anchor=(1, 1))
plt.tight_layout()
# plt.savefig(f"{output_dir}/eventsVsrate-epistatic.pdf")
plt.show()

plt.figure(figsize=(6, 3)) ax = sns.scatterplot(x=dms_df["mutation rate"], y=dms_df["mutation events"], hue=indicator, s=20, alpha=0.7, hue_order=("epistatic background mutation", "epistatically enabled mutation", "other"), palette=("C0", "C1", "lightgrey"), clip_on=False, edgecolors="k", linewidths=0.1) # plt.xlabel("mutation rate") plt.ylabel("mutation events") linthresh_x = 1e-5 linthresh_y = 1.0 plt.xscale("symlog", linthresh=linthresh_x, linscale=.5) plt.yscale("symlog", linthresh=linthresh_y, linscale=.2) plt.xlim(0, None) plt.ylim(0, None) sns.move_legend(ax, "upper left", bbox_to_anchor=(1, 1)) plt.tight_layout() # plt.savefig(f"{output_dir}/eventsVsrate-epistatic.pdf") plt.show()

LBI of nodes with the above mutations

In [67]:

lbi_dat = []
pairs = set((x, y) for x, y in volcano_df.loc[volcano_df[f"FDR < {FDR_tresh}"], ["background mutation", "mutation event"]].values)
for gc, tree in trees.items():
    for node in tree.tree.traverse():
        mutations = set(node.mutations)
        # if node.get_distance(tree.tree) <= 4:
        if len(mutations) == 2:
            if any(set(pair) <= mutations for pair in pairs):            
                lbi_dat.append([node.LBI, node.LBR, "enabling and enabled mutation"])
            elif any(pair[0] in mutations for pair in pairs):
                lbi_dat.append([node.LBI, node.LBR, "enabling mutation only"])
            elif any(pair[1] in mutations for pair in pairs):
                lbi_dat.append([node.LBI, node.LBR, "enabled mutation only"])
            # else:
            #     lbi_dat.append([node.LBI, node.LBR, "none"])

lbi_df = pd.DataFrame(lbi_dat, columns=("LBI", "LBR", "mutation pair on tree node"))

display(lbi_df)

# plt.figure(figsize=(10, 3))
sns.boxplot(data=lbi_df, y="mutation pair on tree node", x="LBI", order=("enabled mutation only", "enabling mutation only", "enabling and enabled mutation"), fliersize=0)
sns.swarmplot(data=lbi_df, y="mutation pair on tree node", x="LBI", order=("enabled mutation only", "enabling mutation only", "enabling and enabled mutation"), color="k", size=3)
plt.tight_layout()
# plt.savefig(f"{output_dir}/epistasis.LBI.pdf")
plt.show()

lbi_dat = [] pairs = set((x, y) for x, y in volcano_df.loc[volcano_df[f"FDR < {FDR_tresh}"], ["background mutation", "mutation event"]].values) for gc, tree in trees.items(): for node in tree.tree.traverse(): mutations = set(node.mutations) # if node.get_distance(tree.tree) <= 4: if len(mutations) == 2: if any(set(pair) <= mutations for pair in pairs): lbi_dat.append([node.LBI, node.LBR, "enabling and enabled mutation"]) elif any(pair[0] in mutations for pair in pairs): lbi_dat.append([node.LBI, node.LBR, "enabling mutation only"]) elif any(pair[1] in mutations for pair in pairs): lbi_dat.append([node.LBI, node.LBR, "enabled mutation only"]) # else: # lbi_dat.append([node.LBI, node.LBR, "none"]) lbi_df = pd.DataFrame(lbi_dat, columns=("LBI", "LBR", "mutation pair on tree node")) display(lbi_df) # plt.figure(figsize=(10, 3)) sns.boxplot(data=lbi_df, y="mutation pair on tree node", x="LBI", order=("enabled mutation only", "enabling mutation only", "enabling and enabled mutation"), fliersize=0) sns.swarmplot(data=lbi_df, y="mutation pair on tree node", x="LBI", order=("enabled mutation only", "enabling mutation only", "enabling and enabled mutation"), color="k", size=3) plt.tight_layout() # plt.savefig(f"{output_dir}/epistasis.LBI.pdf") plt.show()

	LBI	LBR	mutation pair on tree node
0	3.533043	0.000000	enabling and enabled mutation
1	1.927595	0.000000	enabled mutation only
2	9.857638	0.284250	enabled mutation only
3	10.389162	0.368327	enabled mutation only
4	15.610569	1.304021	enabled mutation only
...	...	...	...
686	6.050473	3.251327	enabling and enabled mutation
687	1.566488	0.000000	enabling and enabled mutation
688	3.919638	0.617778	enabling and enabled mutation
689	5.682603	2.183376	enabled mutation only
690	5.454339	0.801159	enabled mutation only

691 rows × 3 columns

/opt/conda/envs/replay/lib/python3.10/site-packages/seaborn/categorical.py:3399: UserWarning: 5.5% of the points cannot be placed; you may want to decrease the size of the markers or use stripplot.
  warnings.warn(msg, UserWarning)

/opt/conda/envs/replay/lib/python3.10/site-packages/seaborn/categorical.py:3399: UserWarning: 8.2% of the points cannot be placed; you may want to decrease the size of the markers or use stripplot.
  warnings.warn(msg, UserWarning)

In [68]:

from scipy.stats import kruskal, mannwhitneyu

kruskal(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI,
        lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI,
        lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

from scipy.stats import kruskal, mannwhitneyu kruskal(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI, lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI, lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

Out[68]:

KruskalResult(statistic=np.float64(14.430264030514346), pvalue=np.float64(0.00073537350683911))

In [69]:

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI,
             lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI)

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI, lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI)

Out[69]:

MannwhitneyuResult(statistic=np.float64(12598.0), pvalue=np.float64(0.01344668091186011))

In [70]:

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI,
             lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabled mutation only"].LBI, lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

Out[70]:

MannwhitneyuResult(statistic=np.float64(24463.0), pvalue=np.float64(0.010099305806099471))

In [71]:

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI,
             lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

mannwhitneyu(lbi_df[lbi_df["mutation pair on tree node"] == "enabling mutation only"].LBI, lbi_df[lbi_df["mutation pair on tree node"] == "enabling and enabled mutation"].LBI)

Out[71]:

MannwhitneyuResult(statistic=np.float64(1138.0), pvalue=np.float64(0.00014983148098383442))

Redoing above using Yun's approach of coincidence of mutations on nodes¶

In [72]:

n_mutations, mutation_counts.sum()

n_mutations, mutation_counts.sum()

Out[72]:

(18898, np.int64(18898))

In [73]:

mutations =  dms_df.index.unique()

n_mutations = 0
mutation_counts = pd.Series(0, index=mutations)

epi_df = pd.DataFrame(np.zeros((len(mutations), len(mutations))), index=mutations, columns=mutations)

for gc, tree in trees.items():
    print(gc, end="       \r")
    for node in tree.tree.iter_descendants(strategy="preorder"):
        if any(mutation.endswith("*") for mutation in node.mutations):
            continue
        for i, mutation1 in enumerate(node.mutations):
            n_mutations += 1
            mutation_counts[mutation1] += 1
            for mutation2 in node.mutations[i + 1:]:
                epi_df.loc[mutation1, mutation2] += 1

volcano_dat = []
                
for i, mutation1 in enumerate(mutations):
    mutation1_total = mutation_counts[mutation1]
    if mutation1_total >= 50:
        for mutation2 in mutations[i + 1:]:
            if mutation1[:-1] != mutation2[:-1] and mutation_counts[mutation2] >= 5:
                print(mutation1, end="       \r")
                table = np.zeros((2, 2), dtype=int)
                table[0, 0] = n_mutations - epi_df.loc[mutation1, mutation2]
                table[0, 1] = mutation_counts[mutation2] - epi_df.loc[mutation1, mutation2]
                table[1, 0] = mutation1_total - epi_df.loc[mutation1, mutation2]
                table[1, 1] = epi_df.loc[mutation1, mutation2]
                
                odds_ratio, p_value = fisher_exact(table, alternative="two-sided")
                row = [mutation1, mutation2, table, odds_ratio, p_value]
                # if p_value < 1e-6:
                #     print(row)
                volcano_dat.append(row)

volcano_df = pd.DataFrame(volcano_dat, columns=["mutation 1", "mutation 2", "contingency table", "odds ratio", "p-value"])

_, volcano_df["FDR"] = fdrcorrection(volcano_df["p-value"], method="p")

FDR_tresh = 0.05
volcano_df[f"FDR < {FDR_tresh}"] = volcano_df.FDR < FDR_tresh

volcano_df[volcano_df[f"FDR < {FDR_tresh}"]].sort_values("p-value")

mutations = dms_df.index.unique() n_mutations = 0 mutation_counts = pd.Series(0, index=mutations) epi_df = pd.DataFrame(np.zeros((len(mutations), len(mutations))), index=mutations, columns=mutations) for gc, tree in trees.items(): print(gc, end=" \r") for node in tree.tree.iter_descendants(strategy="preorder"): if any(mutation.endswith("*") for mutation in node.mutations): continue for i, mutation1 in enumerate(node.mutations): n_mutations += 1 mutation_counts[mutation1] += 1 for mutation2 in node.mutations[i + 1:]: epi_df.loc[mutation1, mutation2] += 1 volcano_dat = [] for i, mutation1 in enumerate(mutations): mutation1_total = mutation_counts[mutation1] if mutation1_total >= 50: for mutation2 in mutations[i + 1:]: if mutation1[:-1] != mutation2[:-1] and mutation_counts[mutation2] >= 5: print(mutation1, end=" \r") table = np.zeros((2, 2), dtype=int) table[0, 0] = n_mutations - epi_df.loc[mutation1, mutation2] table[0, 1] = mutation_counts[mutation2] - epi_df.loc[mutation1, mutation2] table[1, 0] = mutation1_total - epi_df.loc[mutation1, mutation2] table[1, 1] = epi_df.loc[mutation1, mutation2] odds_ratio, p_value = fisher_exact(table, alternative="two-sided") row = [mutation1, mutation2, table, odds_ratio, p_value] # if p_value < 1e-6: # print(row) volcano_dat.append(row) volcano_df = pd.DataFrame(volcano_dat, columns=["mutation 1", "mutation 2", "contingency table", "odds ratio", "p-value"]) _, volcano_df["FDR"] = fdrcorrection(volcano_df["p-value"], method="p") FDR_tresh = 0.05 volcano_df[f"FDR < {FDR_tresh}"] = volcano_df.FDR < FDR_tresh volcano_df[volcano_df[f"FDR < {FDR_tresh}"]].sort_values("p-value")

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K49(H)N       
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K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       

K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
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K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)N       
K49(H)R       
K49(H)R       
K49(H)R       
K49(H)R       
K49(H)R       
K49(H)R       
K49(H)R       
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K49(H)R       

K49(H)R       
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K49(H)R       
K49(H)R       

K49(H)R       
K49(H)R       
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K49(H)R       
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K49(H)T       
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K49(H)T       
K49(H)T       
K49(H)T       
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K49(H)T       

K49(H)T       
K49(H)T       
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K49(H)T       
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S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       

S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       

S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
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S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       

S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
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S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
S57(H)N       
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S57(H)N       
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S57(H)N       
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S57(H)N       
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S57(H)N       

S57(H)N       
S57(H)N       
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S57(H)R       
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Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       

Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       

Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
Y58(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       

S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       

S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       

S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
S59(H)N       
G63(H)D       
G63(H)D       
G63(H)D       
G63(H)D       
G63(H)D       
G63(H)D       
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G63(H)D       
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G63(H)D       
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S64(H)G       
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S64(H)G       
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S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       

S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       

S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)G       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       

S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       

S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       

S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       

S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)N       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
S64(H)R       
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S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       

S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
S64(H)T       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       

T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       

T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)I       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       

T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       

T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       

T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)N       
T65(H)S       
T65(H)S       
T65(H)S       
T65(H)S       
T65(H)S       
T65(H)S       
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T65(H)S       
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T65(H)S       
T65(H)S       
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Y67(H)F       
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K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       

K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
K72(H)T       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       

S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
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S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
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S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       

S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       

S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
S74(H)G       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       

I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
I76(H)F       
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I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
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I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       

I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
I78(H)L       
T79(H)S       
T79(H)S       
T79(H)S       
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T79(H)S       
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K84(H)R       
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K84(H)R       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
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T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       

T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
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T96(H)A       
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T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
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T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)A       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       

T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
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T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
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T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       

T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)I       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       

T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
T96(H)N       
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T96(H)N       
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T96(H)P       
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A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       

A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)T       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       

A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
A105(H)V       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
D107(H)E       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
F115(H)I       
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T123(H)A       
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G36(L)R       
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S69(L)N       
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S69(L)N       
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V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
V71(L)I       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       

D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
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D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)A       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       

D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
D74(L)N       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
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T77(L)A       
T77(L)A       
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T77(L)A       
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T77(L)A       
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T77(L)A       
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T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)A       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
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T77(L)I       
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T77(L)I       
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T77(L)I       
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T77(L)I       
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T77(L)I       
T77(L)I       
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T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       

T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
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T77(L)I       
T77(L)I       
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T77(L)I       
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T77(L)I       
T77(L)I       
T77(L)I       
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T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
T77(L)I       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
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D86(L)E       
D86(L)E       
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D86(L)E       
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D86(L)E       
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D86(L)E       
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D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
D86(L)E       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
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T88(L)I       
T88(L)I       

T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
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T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T88(L)I       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
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T90(L)A       
T90(L)A       
T90(L)A       

T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)A       
T90(L)I       
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T90(L)I       
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T90(L)I       
T90(L)I       
T90(L)I       
T90(L)I       
T90(L)I       
T90(L)I       
T90(L)I       
S92(L)N       
S92(L)N       
S92(L)N       
S92(L)N       
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S92(L)N       
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S92(L)N       
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S92(L)N       
S92(L)N       
S92(L)N       
S92(L)N       
S92(L)N       
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S92(L)R       
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S92(L)R       
S92(L)T       
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S92(L)T       
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N93(L)S       
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Q95(L)H       
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Y114(L)F       
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Out[73]:

	mutation 1	mutation 2	contingency table	odds ratio	p-value	FDR	FDR < 0.05
16536	S57(H)N	A105(H)G	[[52264, 4857], [604, 948]]	16.889078	0.000000	0.000000	True
58132	N108(L)K	S109(L)N	[[52839, 313], [1985, 373]]	31.721855	0.000000	0.000000	True
43463	A105(H)G	S109(L)R	[[52623, 247], [5216, 589]]	24.057825	0.000000	0.000000	True
43439	A105(H)G	Q105(L)H	[[52355, 923], [4948, 857]]	9.824435	0.000000	0.000000	True
43454	A105(H)G	N108(L)K	[[51697, 843], [4290, 1515]]	21.656741	0.000000	0.000000	True
...	...	...	...	...	...	...	...
45873	S7(L)P	R67(L)K	[[53207, 678], [79, 5]]	4.966861	0.004344	0.049470	True
11781	L50(H)F	Q90(H)K	[[53209, 49], [335, 3]]	9.724459	0.004372	0.049779	True
37366	N92(H)K	N108(L)F	[[53210, 13], [355, 2]]	23.059588	0.004389	0.049955	True
23482	T65(H)I	L99(L)S	[[53210, 33], [148, 2]]	21.789517	0.004390	0.049955	True
23262	T65(H)I	V15(L)I	[[53210, 33], [148, 2]]	21.789517	0.004390	0.049955	True

5165 rows × 7 columns

In [74]:

plt.figure(figsize=(4, 4))
ax = sns.scatterplot(data=volcano_df, x="odds ratio", y="p-value", hue=f"FDR < {FDR_tresh}", palette=["lightgrey", "red"])
ax.invert_yaxis()
plt.axvline(1, ls="--", c="k", lw=1)
plt.axhline(1, ls="--", c="k", lw=1)
plt.xscale("log", base=2)
plt.yscale("log")
plt.tight_layout()
plt.show()

plt.figure(figsize=(4, 4)) ax = sns.scatterplot(data=volcano_df, x="odds ratio", y="p-value", hue=f"FDR < {FDR_tresh}", palette=["lightgrey", "red"]) ax.invert_yaxis() plt.axvline(1, ls="--", c="k", lw=1) plt.axhline(1, ls="--", c="k", lw=1) plt.xscale("log", base=2) plt.yscale("log") plt.tight_layout() plt.show()