QC Report

	general
Report generated at	2020-11-05 02:17:29
Title	ER E2
Description	chip seq data for estrogen receptor e2
Pipeline version	v1.3.6
Pipeline type	tf
Genome	hg19
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': False}, 'ctl1': {'paired_end': False}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	ctl1
Total Reads	12652745	3334448
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	11664005	2283199
Mapped Reads (QC-failed)	0	0
% Mapped Reads	92.2	68.5
Paired Reads	0	0
Paired Reads (QC-failed)	0	0
Read1	0	0
Read1 (QC-failed)	0	0
Read2	0	0
Read2 (QC-failed)	0	0
Properly Paired Reads	0	0
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	0.0	0.0
With itself	0	0
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	ctl1
Unpaired Reads	8198782	1401512
Paired Reads	0	0
Unmapped Reads	0	0
Unpaired Duplicate Reads	2292040	411572
Paired Duplicate Reads	0	0
Paired Optical Duplicate Reads	0	0
% Duplicate Reads	27.9559	29.366300000000003

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	ctl1
Total Reads	8198782	1401512
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	8198782	1401512
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	0	0
Paired Reads (QC-failed)	0	0
Read1	0	0
Read1 (QC-failed)	0	0
Read2	0	0
Read2 (QC-failed)	0	0
Properly Paired Reads	0	0
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	0.0	0.0
With itself	0	0
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	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	ctl1
Total Fragments	8168369	1391715
Distinct Fragments	5895750	984761
Positions with Two Read	1134287	210255
NRF = Distinct/Total	0.721778	0.707588
PBC1 = OneRead/Distinct	0.740653	0.701121
PBC2 = OneRead/TwoRead	3.849736	3.283808

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	0	0
N1	14152	6008
Np	0	0
N optimal	14152	6008
N conservative	14152	6008
Optimal Set	rep1-pr1_vs_rep1-pr2	rep1-pr1_vs_rep1-pr2
Conservative Set	rep1-pr1_vs_rep1-pr2	rep1-pr1_vs_rep1-pr2
Rescue Ratio	0.0	0.0
Self Consistency Ratio	1.0	1.0
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
Number of peaks	19874

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

Peak calling statistics

Peak region size

	rep1	idr_opt	overlap_opt
Min size	95.0	95.0	95.0
25 percentile	95.0	159.0	101.0
50 percentile (median)	115.0	197.0	142.0
75 percentile	173.0	252.0	197.0
Max size	815.0	815.0	815.0
Mean	145.69029888296265	216.4046271637816	163.72272470322216

Enrichment / Signal-to-noise ratio

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

	rep1
Number of Subsampled Reads	8168369
Estimated Fragment Length	95
Cross-correlation at Estimated Fragment Length	0.219878791332111
Phantom Peak	40
Cross-correlation at Phantom Peak	0.1491048
Argmin of Cross-correlation	1500
Minimum of Cross-correlation	0.04983539
NSC (Normalized Strand Cross-correlation coeff.)	4.412102
RSC (Relative Strand Cross-correlation coeff.)	1.712948

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
AUC	0.09056210480558827
Synthetic AUC	0.4781008541845002
X-intercept	0.6241263251360383
Synthetic X-intercept	3.6147208522726014e-17
Elbow Point	0.7572248786604352
Synthetic Elbow Point	0.5034707402071257
JS Distance	0.24250408886256314
Synthetic JS Distance	0.3738655815632612
% Genome Enriched	22.540461520309254
Diff. Enrichment	40.89262444737237
CHANCE Divergence	0.38224953959734836

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep1-pr1	rep1-pr2
Fraction of Reads in Peaks	0.16669988298261865	0.21498120086617745	0.21544858736334252

FRiP for overlap peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.15922462629205167

FRiP for IDR peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.14163103739067584

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