2. Sample quality control
[1]:
from pylluminator.samples import Samples
from pylluminator.utils import set_logger
from pylluminator.annotations import Channel
from pylluminator.quality_control import *
import matplotlib.pyplot as plt
import pandas as pd
set_logger('WARNING') # set the verbosity level, can be DEBUG, INFO, WARNING, ERROR
2.1. Load pylluminator Samples
We assume that you have already processed the .idat files according to your preferences and saved them. If not, please refer to notebook 1 - Read data and get beta values before going any further.
[2]:
my_samples = Samples.load('preprocessed_samples')
my_samples
[2]:
Samples object with 6 samples: LNCAP_500_1, PREC_500_1, PREC_500_3, LNCAP_500_3, LNCAP_500_2, PREC_500_2
updated annotation - EPICv2 array - genome version hg38
937,688 probes
2.2. Chose a sample and print QCs
[3]:
sample_name = 'LNCAP_500_3'
detection_stats(my_samples, sample_name)
intensity_stats(my_samples, sample_name)
nb_probes_stats(my_samples, sample_name)
type1_color_channels_stats(my_samples, sample_name)
dye_bias_stats(my_samples, sample_name)
betas_stats(my_samples, sample_name)
===================================================================
| Detection - mask not applied
===================================================================
N. Probes w/ Missing Raw Intensity 2
% Probes w/ Missing Raw Intensity 0.00 %
N. Probes w/ Detection Success 868,918
% Detection Success 92.67 %
N. cg probes 933,252
N. Probes w/ Detection Success cg 865,540
% Detection Success cg 92.74 %
N. ch probes 2,914
N. Probes w/ Detection Success ch 2,554
% Detection Success ch 87.65 %
N. snp probes 65
N. Probes w/ Detection Success snp 65
% Detection Success snp 100.00 %
===================================================================
| Signal intensity - mask not applied
===================================================================
Mean in-band signal intensity 2492.96
Mean in-band signal intensity (M+U) 4982.73
Mean in-band type II signal intensity 2368.62
Mean in-band type I Red signal intensity 3291.55
Mean in-band type I Green signal intensity 3252.46
Mean out-of-band type I Red signal intensity 225.18
Mean out-of-band type I Green signal intensity 253.84
Number of NAs in Methylated signal 4,610
Number of NAs in Unmethylated signal 4,600
Number of NAs in Type 1 Red signal 1,506
Number of NAs in Type 1 Green signal 924
Number of NAs in Type 2 signal 6,780
-- note : these NA values don't count probes that don't appear in .idat files; these are only counted in the `Detection - missing raw intensity` QC line
===================================================================
| Number of probes - mask not applied
===================================================================
Total : 937,688
Type II : 809,393
Type I Green : 45,099
Type I Red : 83,196
CG : 933,252
CH : 2,914
SNP : 65
===================================================================
| LNCAP_500_3 Type I color channel - mask not applied
===================================================================
Green to Green : 45,080
Green to Red : 19
Red to Red : 82,706
Red to Green : 490
===================================================================
| Dye bias - mask not applied
===================================================================
Median Inf type I red channel intensity 6538.87
Median Inf type I green channel intensity 6442.33
Median of top 20 Inf type I red channel intensity 29926.23
Median of top 20 Inf type I green channel intensity 22176.17
Ratio of Red-to-green median intensities 1.01
Ratio of top vs global Red-to-green median intensities 1.33
===================================================================
| Betas - mask not applied
===================================================================
Mean 0.53
Median 0.62
Unmethylated fraction (beta values < 0.3) 36.80 %
Methylated fraction (beta values > 0.7) 45.89 %
Number of NAs 103,061
Fraction of NAs 10.99 %
------ cg probes ------
Mean 0.54
Median 0.62
Unmethylated fraction (beta values < 0.3) 36.58 %
Methylated fraction (beta values > 0.7) 46.05 %
Number of NAs 101,837
Fraction of NAs 10.91 %
------ ch probes ------
Mean 0.04
Median 0.03
Unmethylated fraction (beta values < 0.3) 99.87 %
Methylated fraction (beta values > 0.7) 0.00 %
Number of NAs 524
Fraction of NAs 17.98 %
------ snp probes ------
Mean 0.48
Median 0.50
Unmethylated fraction (beta values < 0.3) 36.92 %
Methylated fraction (beta values > 0.7) 32.31 %
Number of NAs 0
Fraction of NAs 0.00 %
2.3. Plot the number of beads per probe
This will work only if you have kept the .idat data when reading the samples (parameter keep_idat=True)
[4]:
count_beads = my_samples.idata[sample_name][Channel.RED].groupby('n_beads').count()
# don't display the (very) long tail of the graph, stop at the last bin that contains at least 5 probes
five_probes_min = count_beads[count_beads.mean_value > 5]
max_more_than_five_probe = five_probes_min.index.max()
# plot
plt.figure(figsize=(10, 7))
plt.bar(five_probes_min.index, five_probes_min['std_dev'])
plt.xlabel('Number of detected beads per probe')
_ = plt.ylabel('Probes count')
