QC Report

	general
Report generated at	2022-02-19 21:54:04
Title	ATAC-Seq (paired end)
Description	This is a input JSON for paired ended sample.
Pipeline version	v1.9.1
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	92479974	106647586
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	87925846	102066035
Mapped Reads (QC-failed)	0	0
% Mapped Reads	95.1	95.7
Paired Reads	92479974	106647586
Paired Reads (QC-failed)	0	0
Read1	46239987	53323793
Read1 (QC-failed)	0	0
Read2	46239987	53323793
Read2 (QC-failed)	0	0
Properly Paired Reads	83745660	97604250
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	90.60000000000001	91.5
With itself	87116724	101244252
With itself (QC-failed)	0	0
Singletons	809122	821783
Singletons (QC-failed)	0	0
% Singleton	0.8999999999999999	0.8
Diff. Chroms	444531	430520
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	30003024	35713624
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	2458804	2919641
Paired Optical Duplicate Reads	13568	11794
% Duplicate Reads	8.1952	8.1751

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)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	81512971	95686417
Rm = Number of Mitochondrial Reads	17220193	17261758
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.1744114368703914	0.15282901206681737

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	53039522	63671402
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	53039522	63671402
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	53039522	63671402
Paired Reads (QC-failed)	0	0
Read1	26519761	31835701
Read1 (QC-failed)	0	0
Read2	26519761	31835701
Read2 (QC-failed)	0	0
Properly Paired Reads	53039522	63671402
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	53039522	63671402
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

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.43954473906283276	0.4809773939816284
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.2578326641196451	1.4454564919802049
NFR / mono-nuc reads (QC pass)	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

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

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.3092367235134585	0.3437342874906383
Fraction of Reads in blacklist regions	0.0011608701903459838	0.0010475503586366765
Fraction of Reads in promoter regions	0.11423219462649004	0.13258864631251563
Fraction of Reads in enhancer regions	0.29141003570884366	0.3015903277895467

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	27045196	32755067
Distinct Fragments	26520373	31837426
Positions with Two Read	497542	855633
NRF = Distinct/Total	0.980595	0.971985
PBC1 = OneRead/Distinct	0.980787	0.972233
PBC2 = OneRead/TwoRead	52.278658	36.176036

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.

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	143686	94575
N1	107757	60967
N2	124850	76089
Np	149347	97196
N optimal	149347	97196
N conservative	143686	94575
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0393984104227274	1.0277134549299498
Self Consistency Ratio	1.158625425726403	1.2480358226581594
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	199778	202475

Top 300000 raw peaks from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	184.0	192.0	504.0	343.0
50 percentile (median)	277.0	301.0	752.0	559.0
75 percentile	562.0	617.0	1071.0	899.0
Max size	2355.0	2384.0	2607.0	2607.0
Mean	426.2307561393146	453.42989504877147	815.5054734762747	663.5945884416828

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	10.41159454102243	11.814650202894272

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.22167715246434894	0.21307103359551147
Synthetic AUC	0.492457954661429	0.4931228835200539
X-intercept	0.15919419236298132	0.15257243051848476
Synthetic X-intercept	3.429894064542702e-150	6.312166003344691e-181
Elbow Point	0.6638676127468915	0.6939774356267582
Synthetic Elbow Point	0.5138685247749809	0.4940950719166765
Synthetic JS Distance	0.3601752322312237	0.38641571422895205

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	0.20032527819538043	0.23875345166735923	0.19832082203452656	0.24429393766784221	0.19822072296280208	0.24431672619103711	0.23453819112939248	0.22480198255299624	0.22481238259453357

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	0.20499223363187494	0.1683640361615627	0.2129587785737779	0.20702510246598682

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	0.17898290309140213	0.1342217601433135	0.18142253252095816	0.18115785802535503

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