QC Report

	general
Report generated at	2021-03-28 10:18:10
Title	max_l4
Description	chipseq of max_l4
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}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	ctl1
Total Reads	37068399	37978291	39720643
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	35765277	37051603	38989224
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	96.5	97.6	98.2
Paired Reads	0	0	0
Paired Reads (QC-failed)	0	0	0
Read1	0	0	0
Read1 (QC-failed)	0	0	0
Read2	0	0	0
Read2 (QC-failed)	0	0	0
Properly Paired Reads	0	0	0
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	0.0	0.0	0.0
With itself	0	0	0
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

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	30284935	31642031	32809292
Paired Reads	0	0	0
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	1634666	1552486	1053382
Paired Duplicate Reads	0	0	0
Paired Optical Duplicate Reads	0	0	0
% Duplicate Reads	5.397600000000001	4.9064000000000005	3.2106000000000003

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	30284935	31642031	32809292
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	30284935	31642031	32809292
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	0	0	0
Paired Reads (QC-failed)	0	0	0
Read1	0	0	0
Read1 (QC-failed)	0	0	0
Read2	0	0	0
Read2 (QC-failed)	0	0	0
Properly Paired Reads	0	0	0
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	0.0	0.0	0.0
With itself	0	0	0
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	30121763	31481399	32597500
Distinct Fragments	28629641	30068904	31735311
Positions with Two Read	1344142	1238494	759022
NRF = Distinct/Total	0.950464	0.955132	0.97355
PBC1 = OneRead/Distinct	0.950976	0.956433	0.97526
PBC2 = OneRead/TwoRead	20.25538	23.220862	40.776379

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	47092	16811
N1	33188	11236
N2	43413	17634
Np	52010	20439
N optimal	52010	20439
N conservative	47092	16811
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.1044338741187463	1.2158110760811374
Self Consistency Ratio	1.3080932867301434	1.5694197223211108
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	105554	104358

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	145.0	145.0	145.0	145.0
25 percentile	151.0	162.0	325.0	215.0
50 percentile (median)	198.0	225.0	447.0	298.0
75 percentile	295.0	354.0	629.0	446.0
Max size	4829.0	5337.0	5272.0	5272.0
Mean	258.78485893476324	300.7642921481822	510.9165810460394	367.15577773505095

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	15000000	15000000
Estimated Fragment Length	145	145
Cross-correlation at Estimated Fragment Length	0.173561163068517	0.182145134242443
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.1726771	0.1777671
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.1632097	0.1627729
NSC (Normalized Strand Cross-correlation coeff.)	1.063424	1.119014
RSC (Relative Strand Cross-correlation coeff.)	1.093375	1.291979

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	0.27158059378356314	0.27084352743321893
Synthetic AUC	0.4909813997011669	0.49117464101767266
X-intercept	0.11992418517052339	0.11527772779437227
Synthetic X-intercept	1.1832631059436512e-104	2.4104965501244816e-109
Elbow Point	0.5789138006181948	0.6253083976119929
Synthetic Elbow Point	0.49647132583442544	0.5004264442020142
JS Distance	0.09769964443191745	0.1269866887058465
Synthetic JS Distance	0.2800420624384133	0.2899008205873608
% Genome Enriched	32.61817076730525	32.11713711047932
Diff. Enrichment	18.309818618202705	18.362494625848413
CHANCE Divergence	0.15665788459529673	0.15689125188248898

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	0.08569590788291274	0.11679458881763942	0.08218204588578296	0.1045288747574745	0.08219539127937343	0.10465485305462387	0.08379861529143863	0.09901731342635732	0.09916888546320468

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	0.0763849628932249	0.05549085708785573	0.09091492894372045	0.07954158451747821

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	0.04907086841619207	0.032388149421486295	0.061607202141986395	0.0544087368982359

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