This is a notebook where I show you what data is available for analysis, roughly how it is formatted, and how to make a few plots¶

Resources¶

Paper¶

https://science.sciencemag.org/content/366/6464/490

Preprint¶

https://www.biorxiv.org/content/10.1101/675314v1

Github and archived Zenodo of the pipeline to get from raw reads to the processed data available here¶

https://github.com/mjohnson11/TnSeq_Pipeline and https://zenodo.org/record/3402230#.Xc3brpNKhTY (this notebook is essentially a simplified version of the one in these repositories used to make the paper figures)

Requirements if using this locally:¶

I think just a normal anaconda distribution of python should be fine for everything here, which you probably already have: https://www.anaconda.com/distribution/

Importing various useful libraries / setting up plotting¶

In [1]:
import pandas as pd
import numpy as np
from matplotlib import pyplot as pl
import seaborn as sns
sns.set_style("white")
sns.set_style("ticks")
colors = ['#FFB000', '#648FFF']
%matplotlib notebook


In [2]:
## READING DATA
def change_well_format(w):
if '_' in w:
plate = int(w[1:3])
t = 'LK' + str(plate) + '-'
n = int(w.split('_')[1])
lets = ['A', 'B', 'C', 'D', 'E', 'F', 'G', 'H']
l = lets[int(np.floor((n-1)/12))]
return t + l + str(((n-1) % 12) + 1).zfill(2)
else:
return w

def get_geno_matrix(seg_names):
# Data from https://www.nature.com/articles/nature11867, can also be dowloaded here http://genomics-pubs.princeton.edu/YeastCross_BYxRM/
map_genos = {'B': 0, 'R': 1}
for w in d.keys():
if change_well_format(w) in seg_names:
d[change_well_format(w)] = d[w].map(map_genos)
assert len([s for s in seg_names if s in d.columns]) == len(seg_names)
return d[['marker'] + seg_names]

# Reading information of segregant fitness in our focal environment (from Jerison et al. 2017)
seg_to_fit = {i[0]: i[1] for i in x_info.as_matrix(['segregant', 'initial fitness, YPD 30C'])}
# Reading data files containing fitness effect information from the small library experiment (few (~100) mutations in many genetic backgrounds)
# This excludes the neutral controls, a few mutations that were unintentionally included in this library, and a few controls that didn't end up getting good enough coverage to really analyze
tp = tp_all.loc[tp_all['Type']=='Experiment']
# Reading same thing for large library experiment (many mutations in a few genetic backgrounds)
# Reading aggregate data on DFE (distribution of fitness effect) data statistics for each segregant
# For bioinformatic reasons, the BT (large library) data has a longer edge sequence, changing so the two exps are comparable
bt['Long.Edge'] = bt['Edge']
bt['Edge'] = bt['Long.Edge'].str[:15]
# Making a few dictionaries that point from the names I am used to to the dataframes
dats = {'BT': bt, 'TP': tp, 'BT.DFE': bt_dfe, 'TP.DFE': tp_dfe}
exps = {'BT': 'E1', 'TP': 'E2'}
# Getting a list of segregants in each experiment by looking for columns like segregant.mean.s in the dataframe
segs_all = {exp: [i.split('.')[0] for i in dats[exp] if '.mean.s' in i] for exp in exps}
# Getting genotype information on these segregants using data from Bloom et al. 2013
gm = get_geno_matrix(segs_all['TP'])
# Making restricted lists of segregants that have at least 50 mutations with s measured for DFE comparisons
segs_use = {exp: [s for s in segs_all[exp] if len(dats[exp].loc[pd.notnull(dats[exp][s + '.mean.s'])])>=50] for exp in exps}
# Making some
sorted_segs = {exp: sorted(segs_use[exp], key=lambda x: seg_to_fit[x]) for exp in exps}

/anaconda3/lib/python3.7/site-packages/ipykernel_launcher.py:26: FutureWarning: Method .as_matrix will be removed in a future version. Use .values instead.


Let's just take a look at all this data, to see what's what¶

for tp and bt, each row is a mutation for tp_dfe and bt_dfe, each row is a DFE statistic (or background fitness, which we can treat similarly) for gm, each row is a genotyped allele that is different between RM and BY (and takes each state in ~half the segregants)

In [3]:
tp.iloc[:3]

Out[3]:
Edge Type Edge.ID Gene.Use Gene_ORF Gene_ORF.nearby briefDescription briefDescription.nearby chromosome description ... full_model_p full_model_aic full_model_r2_95_conf_low full_model_r2_95_conf_high full_model_p_values full_model_params full_model_coeffs qtls resid.qtls full.model.qtls
0 TGATCATCACGGGAC Experiment 888 in FLC2 FLC2 FLC2 Putative calcium channel involved in calcium r... Putative calcium channel involved in calcium r... chr01 Putative calcium channel involved in calcium r... ... 8.990535e-25 -1049.391200 0.484313 0.664302 3.3295688484889364e-32;1.8707009014831425e-05;... x;locus_9649106_chr14_401893_C_T;locus_9717516... -0.009667588479105496;-0.06737728192983725;0.3... chr14_401893;chr14_374661;chr14_402771 chr14_470303;chr14_420065;chr14_492376 chr14_401893;chr14_374661;chr14_402771|chr14_4...
1 TCGAAAGCACAGTAG Experiment 377 nearby SRB2 NaN SRB2 NaN Subunit of the RNA polymerase II mediator complex chr08 NaN ... 5.640931e-01 -83.796365 0.000082 0.396975 0.00020013790535836575;0.5640930907526316 x -0.049382849134784586;-0.08592702839231847 NaN NaN NaN
2 GTTGAACTGGTTGTT Experiment 197 in STM1 STM1 STM1 Protein required for optimal translation under... Protein required for optimal translation under... chr12 Protein required for optimal translation under... ... 2.500225e-07 -803.596564 0.103969 0.347860 1.4054000123711754e-50;0.9145877127045838;4.10... x;locus_9680363_chr14_433150_G_A -0.028546289780384954;-0.0024826738390553073;0... chr14_433150;chr14_420065;chr14_485550 chr14_433150;chr14_420065;chr14_492376 chr14_433150;chr14_420065;chr14_485550

3 rows × 1696 columns

In [4]:
bt.iloc[:3]

Out[4]:
Edge.ID Edge Gene.Use Gene_ORF Gene_ORF.nearby briefDescription briefDescription.nearby chromosome description description.nearby ... full_model_r2 full_model_p full_model_aic full_model_p_values full_model_params full_model_coeffs qtls resid.qtls full.model.qtls Long.Edge
0 0 TCTCCAAGGGATACT in YMR085W YMR085W YMR085W Putative protein of unknown function Putative protein of unknown function chr13 Putative protein of unknown function; YMR085W ... Putative protein of unknown function; YMR085W ... ... 0.010223 0.781072 -80.547553 0.26814250806982043;0.7810722126668491 x 0.001490528225059142;0.006953626038516742 NaN NaN NaN TCTCCAAGGGATACTTAACGTTATTCCTTT
1 1 TGTGTCGATTTAGTG in RKM3 RKM3 RKM3 Ribosomal lysine methyltransferase Ribosomal lysine methyltransferase chr02 Ribosomal lysine methyltransferase; specific f... Ribosomal lysine methyltransferase; specific f... ... 0.030625 0.567432 -114.446981 0.15944797128735522;0.5674322326468317 x 0.001162120166529229;-0.009006462611979207 NaN NaN NaN TGTGTCGATTTAGTGTTAAAGAATGACGTC
2 2 TATGGTGCAGAAAAG nearby YNL143C NaN MEP2|YNL143C NaN Ammonium permease involved in regulation of ps... chr14 NaN Ammonium permease involved in regulation of ps... ... 0.144110 0.132863 -171.801874 0.10689612722798757;0.13286265303302433 x 0.0006108064927804045;0.012645873311052722 NaN NaN NaN TATGGTGCAGAAAAGTGGCTCGGAATGAAC

3 rows × 246 columns

In [5]:
tp_dfe.iloc[:3]

Out[5]:
DFE.statistic LK4-G06 LK3-G08 LK2-G07 LK4-H03 LK4-D11 LK3-E06 LK1-D12 LK6-C05 LK4-C09 ... full_resid_seg_model_r2 full_resid_seg_model_p full_resid_seg_model_r2_95_conf_low full_resid_seg_model_r2_95_conf_high full_resid_seg_model_p_values full_resid_seg_model_params full_resid_seg_model_coeffs qtls resid.qtls full.model.qtls
0 background.fitness 0.017536 0.093806 -0.019195 0.022961 -0.060829 0.032745 -0.057404 -0.059662 -0.087799 ... NaN NaN NaN NaN NaN NaN NaN chr14_381891;chr14_368185;chr14_393024|chr15_1... NaN chr14_381891;chr14_368185;chr14_393024|chr15_1...
1 mean -0.021606 -0.038078 -0.022692 -0.033220 -0.011229 -0.026932 -0.010239 -0.010303 -0.016691 ... NaN NaN NaN NaN NaN NaN NaN chr14_381891;chr14_371959;chr14_393024|chr04_4... chr14_459667;chr14_414983;chr14_481897 chr14_381891;chr14_371959;chr14_393024|chr04_4...
2 median -0.017210 -0.022665 -0.012889 -0.025198 -0.012115 -0.016404 -0.010974 -0.009748 -0.010328 ... NaN NaN NaN NaN NaN NaN NaN chr14_376315;chr14_368185;chr14_393024 chr14_433150;chr14_414983;chr14_485550 chr14_376315;chr14_368185;chr14_393024|chr14_4...

3 rows × 213 columns

In [6]:
bt_dfe.iloc[:3]

Out[6]:
DFE.statistic LK4-A04 LK3-D08 LK3-G02 LK2-F11 LK1-E09 LK4-E02 LK1-C09 LK2-A12 LK4-H11 ... full_resid_seg_model_r2 full_resid_seg_model_p full_resid_seg_model_r2_95_conf_low full_resid_seg_model_r2_95_conf_high full_resid_seg_model_p_values full_resid_seg_model_params full_resid_seg_model_coeffs qtls resid.qtls full.model.qtls
0 background.fitness 0.036559 0.023625 -0.021251 0.035349 0.001306 -0.021838 0.097835 -0.029612 -0.043350 ... NaN NaN NaN NaN NaN NaN NaN NaN NaN NaN
1 mean -0.005015 -0.005416 -0.003760 -0.004865 -0.002509 -0.003774 -0.003754 -0.002583 -0.003060 ... NaN NaN NaN NaN NaN NaN NaN NaN NaN NaN
2 median 0.001086 0.000173 0.000602 0.000504 0.000646 0.000713 0.000439 0.000787 0.000182 ... NaN NaN NaN NaN NaN NaN NaN NaN NaN NaN

3 rows × 85 columns

In [7]:
gm.iloc[:3]

Out[7]:
marker LK4-G06 LK3-G08 LK2-G07 LK4-A04 LK4-H03 LK4-D11 LK3-E06 LK1-D10 LK1-D12 ... LK2-D09 LK1-D06 LK3-A01 LK2-D05 LK1-C11 LK1-G03 LK3-D04 LK3-C06 LK2-A01 LK2-G10
0 27915_chr01_27915_T_C 1 0 1 1 0 0 0 1 1 ... 0 0 1 0 1 0 1 1 1 1
1 28323_chr01_28323_G_A 1 0 1 1 0 0 0 1 1 ... 0 0 1 0 1 0 1 1 1 1
2 28652_chr01_28652_G_T 1 0 1 1 0 0 0 1 1 ... 0 0 1 0 1 0 1 1 1 1

3 rows × 163 columns

Here are some basic statistics on the data from the two experiments:¶

In [8]:
tp_seg_edges_measured = [len(tp.loc[pd.notnull(tp[s+'.mean.s'])]) for s in segs_all['TP']]
bt_seg_edges_measured = [len(bt.loc[pd.notnull(bt[s+'.mean.s'])]) for s in segs_all['BT']]

In [9]:
print('BT. Tried 20 segregants.')
print(len(segs_all['BT']), ' segregants included.', np.mean(bt_seg_edges_measured), 'mutations measured on average,', 'min:', np.min(bt_seg_edges_measured), 'max:', np.max(bt_seg_edges_measured))
print(len(bt), 'mutations.', len(bt.loc[bt['num.measured']>0]), 'measured in at least one seg.', 'of those, they are measured in', np.mean(bt.loc[bt['num.measured']>0]['num.measured']), 'on average.')
print('Measured in', np.nanmean(bt.loc[bt['num.measured']>0]['num.measured']), 'segs on average')
print(len(bt.loc[bt['num.sig']>0]), 'significant in at least one seg,', len(bt.loc[bt['num.measured']>0].loc[bt['num.sig']==0]), 'in none,',
len(bt.loc[bt['num.measured']>0].loc[bt['num.sig']==0])/len(bt.loc[bt['num.measured']>0]), 'percent')

BT. Tried 20 segregants.
18  segregants included. 413.8888888888889 mutations measured on average, min: 180 max: 614
996 mutations. 710 measured in at least one seg. of those, they are measured in 10.492957746478874 on average.
Measured in 10.492957746478874 segs on average
253 significant in at least one seg, 457 in none, 0.643661971830986 percent

In [10]:
print('TP. Tried 176 segregants.')
print(len(segs_all['TP']), ' segregants included.', np.mean(tp_seg_edges_measured), 'mutations measured on average,', 'min:', np.min(tp_seg_edges_measured), 'max:', np.max(tp_seg_edges_measured))
print(len(tp), 'mutations.', 'Measured in', np.nanmean(tp['num.measured']), 'segs on average')
print(np.min(tp['num.measured']),'is the minimum # segs w measurements for one mutation, and the min # sig effects is:', np.min(tp['num.sig']))

TP. Tried 176 segregants.
162  segregants included. 65.70987654320987 mutations measured on average, min: 1 max: 82
91 mutations. Measured in 116.97802197802197 segs on average
9 is the minimum # segs w measurements for one mutation, and the min # sig effects is: 0


Additional columns in these datasets describe the results of the modeling described in the paper, in terms of the R^2, AIC, etc. for various models of epistasis, the models are background fitness (x), QTLs (qtl), full (both), or resid versions (resid_qtl is the qtl model after regressing out x effects), and the columns like:¶

In [11]:
print([i for i in tp if 'aic' in i or i[-2:] in ['r2', '_p']])

['x_model_r2', 'x_model_p', 'x_model_aic', 'qtl_model_r2', 'qtl_model_p', 'qtl_model_aic', 'resid_qtl_model_r2', 'resid_qtl_model_p', 'resid_qtl_model_aic', 'resid_x_model_r2', 'resid_x_model_p', 'resid_x_model_aic', 'full_model_r2', 'full_model_p', 'full_model_aic']


Plot fitness effect vs. background fitness for a particular mutation¶

In [12]:
def plot_simple(sub, df_row, segs):
measured = [seg for seg in segs if pd.notnull(df_row[seg + '.mean.s'])]
xs = [seg_to_fit[measured[s]] for s in range(len(measured))]
ys = [df_row[measured[s] + '.mean.s'] for s in range(len(measured))]
ye = [df_row[measured[s] + '.stderr.s'] for s in range(len(measured))]
sub.axhline(y=0, xmin=0, xmax=1, color='#333333', linestyle='dashed', alpha=0.5, lw=1)
sub.errorbar(x=xs, y=ys, yerr=ye, marker='o', c='k', linestyle='')
sub.set_xlim([-0.16, 0.12])
sub.set_ylim([-0.2, 0.08])
sub.set_xlabel('X')
sub.set_ylabel('S')
sns.despine()

f, sub = pl.subplots(1,1)
dataframe_row = tp[tp['Gene.Use']=='in RPL16A'].iloc[0]
plot_simple(sub, dataframe_row, segs_use['TP'])


Plot a couple DFEs¶

Note in the paper we plot combined DFEs by background fitness quartiles to make them less noisey, which is why this is different:

In [13]:
def get_dfe(df, segname):
use_df = df.loc[pd.notnull(df[segname + '.mean.s'])]
return list(use_df[segname + '.mean.s'])

def simple_plot_dfe_compare(exp):
f, sub = pl.subplots(1, 1, figsize=(6, 4))
lowest_fit_dfe = get_dfe(dats[exp], sorted_segs[exp][0])
highest_fit_dfe = get_dfe(dats[exp], sorted_segs[exp][-1])
bin_lefts = [(-16.15+i)*0.015-0.005 for i in range(22)]
sub.hist(lowest_fit_dfe, bins=bin_lefts, label='Least Fit Seg', histtype="step", color="r", weights=np.ones_like(lowest_fit_dfe)/float(len(lowest_fit_dfe)))
sub.hist(highest_fit_dfe, bins=bin_lefts, label='Most Fit Seg', histtype="step", color="b", weights=np.ones_like(highest_fit_dfe)/float(len(highest_fit_dfe)))
sub.legend(loc='upper left')
sns.despine()

simple_plot_dfe_compare('BT')
simple_plot_dfe_compare('TP')


OK that concludes the jump-start!¶

If you want to get deeper into the plotting / data exploration you can also check out the (much longer but less well commented) jupyter notebook on https://github.com/mjohnson11/TnSeq_Pipeline

Please email me if you have questions: [email protected]