QC Report

	general
Report generated at	2021-06-18 11:41:14
Title	e2f3_mk562
Description	chipseq of e2f3_mk562
Pipeline version	v1.3.6
Pipeline type	tf
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}, 'ctl1': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	ctl1
Total Reads	70630978	90453580	80880754
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	67864451	87285325	79344211
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	96.1	96.5	98.1
Paired Reads	70630978	90453580	80880754
Paired Reads (QC-failed)	0	0	0
Read1	35315489	45226790	40440377
Read1 (QC-failed)	0	0	0
Read2	35315489	45226790	40440377
Read2 (QC-failed)	0	0	0
Properly Paired Reads	67227454	84933404	78032470
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	95.19999999999999	93.89999999999999	96.5
With itself	67475388	86798368	78921270
With itself (QC-failed)	0	0	0
Singletons	389063	486957	422941
Singletons (QC-failed)	0	0	0
% Singleton	0.6	0.5	0.5
Diff. Chroms	45769	65071	81930
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	29726800	37590505	34284776
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	5463875	7474645	5362598
Paired Optical Duplicate Reads	0	0	0
% Duplicate Reads	18.3803	19.8844	15.6413

Filtered out (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

SAMstat (filtered/deduped BAM)

	rep1	rep2	ctl1
Total Reads	48525850	60231720	57844356
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	48525850	60231720	57844356
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	48525850	60231720	57844356
Paired Reads (QC-failed)	0	0	0
Read1	24262925	30115860	28922178
Read1 (QC-failed)	0	0	0
Read2	24262925	30115860	28922178
Read2 (QC-failed)	0	0	0
Properly Paired Reads	48525850	60231720	57844356
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	48525850	60231720	57844356
With itself (QC-failed)	0	0	0
Singletons	0	0	0
Singletons (QC-failed)	0	0	0
% Singleton	0.0	0.0	0.0
Diff. Chroms	0	0	0
Diff. Chroms (QC-failed)	0	0	0

Filtered and duplicates removed

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2	ctl1
Total Fragments	29491174	37310188	33491508
Distinct Fragments	24079014	29903931	28369316
Positions with Two Read	3523904	4685129	3569796
NRF = Distinct/Total	0.816482	0.801495	0.84706
PBC1 = OneRead/Distinct	0.819649	0.804056	0.850196
PBC2 = OneRead/TwoRead	5.600701	5.132076	6.756543

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1. See more details at the ENCODE portal standard for ChIP-Seq pipeline

NRF (non redundant fraction)
PBC1 (PCR Bottleneck coefficient 1)
PBC2 (PCR Bottleneck coefficient 2)
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	7045	1499
N1	8825	1026
N2	6423	1061
Np	8571	1577
N optimal	8571	1577
N conservative	7045	1499
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.2166075230660043	1.0520346897931954
Self Consistency Ratio	1.373968550521563	1.03411306042885
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	95264	50197

Top 500000 raw peaks from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	175.0	165.0	173.0	170.0
25 percentile	185.0	175.0	291.0	211.0
50 percentile (median)	221.0	207.0	361.0	263.0
75 percentile	280.0	262.0	466.0	349.0
Max size	1620.0	1681.0	1458.0	1527.0
Mean	249.29565208263352	233.1741339123852	402.571972098922	300.5202426788006

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (trimmed/filtered SE BAM)

	rep1	rep2
Number of Subsampled Reads	15000000	15000000
Estimated Fragment Length	175	165
Cross-correlation at Estimated Fragment Length	0.150709606548401	0.152227014337998
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.15147	0.1533155
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.1472979	0.1490438
NSC (Normalized Strand Cross-correlation coeff.)	1.023162	1.021357
RSC (Relative Strand Cross-correlation coeff.)	0.8177449	0.7451787

Performed on subsampled (15000000) reads mapped from FASTQs that are trimmed to 50. Such FASTQ trimming and subsampling reads are for cross-corrleation analysis only. Untrimmed FASTQs are used for all the other analyses.

NOTE1: For SE datasets, reads from replicates are randomly subsampled to 15000000.
NOTE2: For PE datasets, the first end (R1) of each read-pair is selected and trimmed to 50 the reads are then randomly subsampled to 15000000.

Normalized strand cross-correlation coefficient (NSC) = col9 in outFile
Relative strand cross-correlation coefficient (RSC) = col10 in outFile
Estimated fragment length = col3 in outFile, take the top value

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.27157906891822314	0.28641727835734426
Synthetic AUC	0.49497039618079214	0.49548509035986477
X-intercept	0.11927106241752969	0.10597384941421088
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5886620816506057	0.5778039905633972
Synthetic Elbow Point	0.5068659144446912	0.4962758384860509
JS Distance	0.043843125962185155	0.047137371631742156
Synthetic JS Distance	0.2878353258395474	0.27111833664089574
% Genome Enriched	42.68123475548803	44.91583030109161
Diff. Enrichment	11.076918456301964	8.72040286432359
CHANCE Divergence	0.10214943929315666	0.08117362460656223

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.046824733621358514	0.025206187038988758	0.0640301586049432	0.04602767445458971	0.06409326427433067	0.04639661626797309	0.012902927124980817	0.04599878342263838	0.04609731618860768

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.007926271247141693	0.009509962216014764	0.00684224192833942	0.00858491045726748

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.003471721554646725	0.0029890666521039818	0.0026908413042164496	0.003569783694137337

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates