QC Report

	general
Report generated at	2023-09-19 04:44:54
Title	Sample1
Description	ATAT-seq pipeline for Sample1
Pipeline version	v2.2.2
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1
Total Reads	301832452
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	295967238
Mapped Reads (QC-failed)	0
% Mapped Reads	98.1
Paired Reads	301832452
Paired Reads (QC-failed)	0
Read1	150916226
Read1 (QC-failed)	0
Read2	150916226
Read2 (QC-failed)	0
Properly Paired Reads	291586462
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	96.6
With itself	292982866
With itself (QC-failed)	0
Singletons	2984372
Singletons (QC-failed)	0
% Singleton	1.0
Diff. Chroms	130212
Diff. Chroms (QC-failed)	0

Marking duplicates (filtered BAM)

	rep1
Unpaired Reads	0
Paired Reads	124451142
Unmapped Reads	0
Unpaired Duplicate Reads	0
Paired Duplicate Reads	26280939
Paired Optical Duplicate Reads	2098188
% Duplicate Reads	21.1175

Filtered with samtools flag 1804 (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
Rn = Number of Non-mitochondrial Reads	283053878
Rm = Number of Mitochondrial Reads	33048585
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.10455022933497357

SAMstat (filtered/deduped BAM)

	rep1
Total Reads	192995374
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	192995374
Mapped Reads (QC-failed)	0
% Mapped Reads	100.0
Paired Reads	192995374
Paired Reads (QC-failed)	0
Read1	96497687
Read1 (QC-failed)	0
Read2	96497687
Read2 (QC-failed)	0
Properly Paired Reads	192995374
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	100.0
With itself	192995374
With itself (QC-failed)	0
Singletons	0
Singletons (QC-failed)	0
% Singleton	0.0
Diff. Chroms	0
Diff. Chroms (QC-failed)	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1
Fraction of reads in NFR	0.6673164616005597
Fraction of reads in NFR (QC pass)	True
Fraction of reads in NFR (QC reason)	OK
NFR / mono-nuc reads	3.341204299955334
NFR / mono-nuc reads (QC pass)	True
NFR / mono-nuc reads (QC reason)	OK
Presence of NFR peak	True
Presence of Mono-Nuc peak	True
Presence of Di-Nuc peak	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
Fraction of Reads in universal DHS regions	0.47295425847875505
Fraction of Reads in blacklist regions	0.001803110576111529
Fraction of Reads in promoter regions	0.08944977613815759
Fraction of Reads in enhancer regions	0.381499206297038

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
Total Fragments	111147804
Distinct Fragments	97130901
Positions with Two Read	9613030
NRF = Distinct/Total	0.87389
PBC1 = OneRead/Distinct	0.888055
PBC2 = OneRead/TwoRead	8.972982

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.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
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	167259	82753
Np	0	0
N optimal	167259	82753
N conservative	167259	82753
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	284392

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	idr_opt	overlap_opt
Min size	150.0	150.0	150.0
25 percentile	211.0	493.0	336.5
50 percentile (median)	330.0	741.0	512.0
75 percentile	587.0	1045.0	806.0
Max size	3209.0	3209.0	3209.0
Mean	458.2253122450702	803.2245114980726	617.3681236884114

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1
TSS enrichment	14.438162213465061

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
AUC	0.2741197747444691
Synthetic AUC	0.4974801207911718
X-intercept	0.1278728909471192
Synthetic X-intercept	0.0
Elbow Point	0.5959316624875527
Synthetic Elbow Point	0.4982102715343964
Synthetic JS Distance	0.29790438688271353

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep1-pr1	rep1-pr2
Fraction of Reads in Peaks	0.19405741818454156	0.17633236974548033	0.18052367597705918

FRiP for overlap peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.1594157018499314

FRiP for IDR peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.11851859723850168

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