QC Report

	general
Report generated at	2025-03-12 10:17:35
Title	mut22
Description	ATAC-seq on mut22
Pipeline version	v2.2.3
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	199900
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	170810
Mapped Reads (QC-failed)	0
% Mapped Reads	85.39999999999999
Paired Reads	199900
Paired Reads (QC-failed)	0
Read1	99950
Read1 (QC-failed)	0
Read2	99950
Read2 (QC-failed)	0
Properly Paired Reads	136554
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	68.30000000000001
With itself	152798
With itself (QC-failed)	0
Singletons	18012
Singletons (QC-failed)	0
% Singleton	9.0
Diff. Chroms	3237
Diff. Chroms (QC-failed)	0

Marking duplicates (filtered BAM)

	rep1
Unpaired Reads	0
Paired Reads	62030
Unmapped Reads	0
Unpaired Duplicate Reads	0
Paired Duplicate Reads	10663
Paired Optical Duplicate Reads	0
% Duplicate Reads	17.1901

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	156218
Rm = Number of Mitochondrial Reads	45589
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.2259039577418028

SAMstat (filtered/deduped BAM)

	rep1
Total Reads	76994
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	76994
Mapped Reads (QC-failed)	0
% Mapped Reads	100.0
Paired Reads	76994
Paired Reads (QC-failed)	0
Read1	38497
Read1 (QC-failed)	0
Read2	38497
Read2 (QC-failed)	0
Properly Paired Reads	76994
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	100.0
With itself	76994
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	0.4596344561921154
Fraction of reads in NFR (QC pass)	True
Fraction of reads in NFR (QC reason)	OK
NFR / mono-nuc reads	0.9854155684413987
NFR / mono-nuc reads (QC pass)	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]
Presence of NFR peak	False
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	0.5674727900875393
Fraction of Reads in blacklist regions	0.16223342078603528
Fraction of Reads in promoter regions	0.16180481595968518
Fraction of Reads in enhancer regions	0.2666181780398473

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	47130
Distinct Fragments	38497
Positions with Two Read	1491
NRF = Distinct/Total	0.816826
PBC1 = OneRead/Distinct	0.936255
PBC2 = OneRead/TwoRead	24.173709

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	1945	410
Np	0	0
N optimal	1945	410
N conservative	1945	410
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	16269

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	152.0	150.0
25 percentile	150.0	276.0	225.0
50 percentile (median)	230.0	358.5	319.0
75 percentile	297.0	422.25	390.0
Max size	1871.0	1871.0	1871.0
Mean	237.84491978609626	386.109756097561	329.58817480719796

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (filtered BAM)

	rep1
Number of Subsampled Reads	47130
Estimated Fragment Length	0
Cross-correlation at Estimated Fragment Length	0.107659125224664
Phantom Peak	50
Cross-correlation at Phantom Peak	0.1589028
Argmin of Cross-correlation	1500
Minimum of Cross-correlation	0.03825651
NSC (Normalized Strand Cross-correlation coeff.)	2.814139
RSC (Relative Strand Cross-correlation coeff.)	0.5752571

Performed on subsampled (25000000) reads. Such FASTQ trimming is for cross-corrleation analysis only.

Fragment = read (for single-ended dataset) or pair of reads (for paired-ended dataset)
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

TSS enrichment (filtered/deduped BAM)

	rep1
TSS enrichment	27.37608995573771

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	0.0008008946743672195
Synthetic AUC	0.24870292914761533
X-intercept	0.9966866494167144
Synthetic X-intercept	0.929075546053107
Elbow Point	0.9966846498086375
Synthetic Elbow Point	0.6478416921898045
Synthetic JS Distance	0.5886855341817002

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep1-pr1	rep1-pr2
Fraction of Reads in Peaks	0.7369145647712809	0.7452854693750325	0.7468568162926018

FRiP for overlap peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.3966672727745019

FRiP for IDR peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.27567083149336313

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