QC Report

	general
Report generated at	2022-02-19 21:12:27
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	95141212	99125044
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	91210544	94426065
Mapped Reads (QC-failed)	0	0
% Mapped Reads	95.89999999999999	95.3
Paired Reads	95141212	99125044
Paired Reads (QC-failed)	0	0
Read1	47570606	49562522
Read1 (QC-failed)	0	0
Read2	47570606	49562522
Read2 (QC-failed)	0	0
Properly Paired Reads	86814690	89890386
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	91.2	90.7
With itself	90271218	93643450
With itself (QC-failed)	0	0
Singletons	939326	782615
Singletons (QC-failed)	0	0
% Singleton	1.0	0.8
Diff. Chroms	488190	433285
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	30193371	32119048
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	3189320	2836208
Paired Optical Duplicate Reads	11675	11634
% Duplicate Reads	10.563	8.830300000000001

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	82770312	87675036
Rm = Number of Mitochondrial Reads	22369180	18170274
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.21275716264636318	0.1716682014536119

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	51672938	56715912
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	51672938	56715912
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	51672938	56715912
Paired Reads (QC-failed)	0	0
Read1	25836469	28357956
Read1 (QC-failed)	0	0
Read2	25836469	28357956
Read2 (QC-failed)	0	0
Properly Paired Reads	51672938	56715912
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	51672938	56715912
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	0.4415514128681283	0.5401171810582639
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.26577304894548	1.7391794670866596
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	0.30288701602374535	0.29481627660329257
Fraction of Reads in blacklist regions	0.0012302764746993872	0.001145939432306052
Fraction of Reads in promoter regions	0.10955686320758459	0.10084254662077902
Fraction of Reads in enhancer regions	0.2872347030083716	0.2874315412577691

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	26296685	28986428
Distinct Fragments	25836941	28359324
Positions with Two Read	435106	590558
NRF = Distinct/Total	0.982517	0.978366
PBC1 = OneRead/Distinct	0.982763	0.978614
PBC2 = OneRead/TwoRead	58.357226	46.994275

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	128857	83464
N1	99290	58329
N2	100260	59532
Np	126494	81896
N optimal	128857	83464
N conservative	128857	83464
Optimal Set	rep1_vs_rep2	rep1_vs_rep2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.018680727939665	1.0191462342483149
Self Consistency Ratio	1.0097693624735622	1.0206243892403435
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	220510	209515

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	172.0	174.0	470.0	327.0
50 percentile (median)	247.0	248.0	705.0	525.0
75 percentile	471.0	458.0	1022.0	853.0
Max size	2392.0	2129.0	2672.0	2672.0
Mean	381.49718380118816	372.5716631267451	770.8509057797373	631.3261832884515

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	10.524858979821033	10.000067863009896

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	0.22565840415716593	0.2329110617931964
Synthetic AUC	0.4923598555209024	0.49270478971836684
X-intercept	0.15949104897174138	0.15428077878144456
Synthetic X-intercept	2.525332319494661e-146	1.2755926707974974e-160
Elbow Point	0.6590751870379035	0.6507979350855516
Synthetic Elbow Point	0.5125373010911483	0.511739268100284
Synthetic JS Distance	0.3534204835405633	0.3439116004619953

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	0.20078455767310927	0.19048978706363745	0.19148513709496692	0.1936787686672481	0.19174327543532654	0.19327232188384805	0.2030644388237351	0.1904901991212159	0.19047992834096733

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	0.17210087568970425	0.1627720877802613	0.15628975515724758	0.1715277263297839

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	0.14942429963967696	0.13222439567883676	0.1290612235945355	0.14839091843856633

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