QC Report

	general
Report generated at	2021-03-29 07:00:23
Title	max_a549_2
Description	chipseq of max_a549_2
Pipeline version	v1.3.6
Pipeline type	tf
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': False}, 'rep2': {'paired_end': False}, 'ctl1': {'paired_end': False}, 'ctl2': {'paired_end': False}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	ctl1	ctl2
Total Reads	28521681	32653391	24114469	28739630
Total Reads (QC-failed)	0	0	0	0
Duplicate Reads	0	0	0	0
Duplicate Reads (QC-failed)	0	0	0	0
Mapped Reads	27756105	32325948	23866522	27471733
Mapped Reads (QC-failed)	0	0	0	0
% Mapped Reads	97.3	99.0	99.0	95.6
Paired Reads	0	0	0	0
Paired Reads (QC-failed)	0	0	0	0
Read1	0	0	0	0
Read1 (QC-failed)	0	0	0	0
Read2	0	0	0	0
Read2 (QC-failed)	0	0	0	0
Properly Paired Reads	0	0	0	0
Properly Paired Reads (QC-failed)	0	0	0	0
% Properly Paired Reads	0.0	0.0	0.0	0.0
With itself	0	0	0	0
With itself (QC-failed)	0	0	0	0
Singletons	0	0	0	0
Singletons (QC-failed)	0	0	0	0
% Singleton	0.0	0.0	0.0	0.0
Diff. Chroms	0	0	0	0
Diff. Chroms (QC-failed)	0	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1	ctl2
Unpaired Reads	22602242	26570655	19636299	22191270
Paired Reads	0	0	0	0
Unmapped Reads	0	0	0	0
Unpaired Duplicate Reads	855003	1753602	3255994	1007565
Paired Duplicate Reads	0	0	0	0
Paired Optical Duplicate Reads	0	0	0	0
% Duplicate Reads	3.7828	6.5998	16.5815	4.5404

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	ctl2
Total Reads	22602242	26570655	19636299	22191270
Total Reads (QC-failed)	0	0	0	0
Duplicate Reads	0	0	0	0
Duplicate Reads (QC-failed)	0	0	0	0
Mapped Reads	22602242	26570655	19636299	22191270
Mapped Reads (QC-failed)	0	0	0	0
% Mapped Reads	100.0	100.0	100.0	100.0
Paired Reads	0	0	0	0
Paired Reads (QC-failed)	0	0	0	0
Read1	0	0	0	0
Read1 (QC-failed)	0	0	0	0
Read2	0	0	0	0
Read2 (QC-failed)	0	0	0	0
Properly Paired Reads	0	0	0	0
Properly Paired Reads (QC-failed)	0	0	0	0
% Properly Paired Reads	0.0	0.0	0.0	0.0
With itself	0	0	0	0
With itself (QC-failed)	0	0	0	0
Singletons	0	0	0	0
Singletons (QC-failed)	0	0	0	0
% Singleton	0.0	0.0	0.0	0.0
Diff. Chroms	0	0	0	0
Diff. Chroms (QC-failed)	0	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	ctl2
Total Fragments	22449016	26421985	19514491	22021952
Distinct Fragments	21725704	24795583	16359396	21162204
Positions with Two Read	660578	1452615	2406024	783699
NRF = Distinct/Total	0.96778	0.938445	0.83832	0.96096
PBC1 = OneRead/Distinct	0.968444	0.938219	0.83155	0.961553
PBC2 = OneRead/TwoRead	31.851076	16.015047	5.653998	25.964791

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	25930	8538
N1	25309	9590
N2	20839	6541
Np	25375	8608
N optimal	25930	8608
N conservative	25930	8538
Optimal Set	rep1_vs_rep2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.021871921182266	1.008198641368002
Self Consistency Ratio	1.2145016555496906	1.466136676349182
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	154854	117363

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	95.0	125.0	110.0	110.0
25 percentile	95.0	125.0	270.0	177.0
50 percentile (median)	97.0	139.0	375.0	249.0
75 percentile	137.0	191.0	542.0	371.0
Max size	1606.0	1798.0	1773.0	1793.0
Mean	132.86121120539346	182.9050552559154	432.2259526022305	304.75098341689164

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	15000000	15000000
Estimated Fragment Length	95	125
Cross-correlation at Estimated Fragment Length	0.177462010013098	0.175525985550464
Phantom Peak	35	35
Cross-correlation at Phantom Peak	0.1756144	0.1732962
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.1669802	0.1655691
NSC (Normalized Strand Cross-correlation coeff.)	1.062772	1.060137
RSC (Relative Strand Cross-correlation coeff.)	1.21399	1.288566

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	0.26846505503782797	0.26590975740749534
Synthetic AUC	0.48771222116725216	0.4886669718907847
X-intercept	0.14494129972329298	0.1394391484461221
Synthetic X-intercept	6.86095071025341e-56	7.120729866361571e-66
Elbow Point	0.5690108123670446	0.6004502487164313
Synthetic Elbow Point	0.49159848835299685	0.5194927694559459
JS Distance	0.06844666440378379	0.06964538411477579
Synthetic JS Distance	0.2620486746478402	0.27132065406713574
% Genome Enriched	32.337534588378304	36.77464371971018
Diff. Enrichment	17.715258725865752	17.027380822598797
CHANCE Divergence	0.15172011253468756	0.14876453042265597

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	0.06677713653362352	0.06463084933359753	0.08887560800384316	0.06612347094478963	0.08887649287181333	0.06595185801599013	0.045275652561206636	0.055035560442258255	0.05518080529566532

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	0.03561140601498423	0.031243581942003808	0.03300088010626761	0.035502789270276267

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	0.02230263146789989	0.02216589840954716	0.019439076680646376	0.022373137787671936

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