QC Report

	general
Report generated at	2021-06-14 22:52:43
Title	lung
Description	ATAC-seq on postnatal p0 mouse lung
Pipeline version	v1.10.0
Pipeline type	atac
Genome	mm10
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	128292886	103794214
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	122132536	98021079
Mapped Reads (QC-failed)	0	0
% Mapped Reads	95.19999999999999	94.39999999999999
Paired Reads	128292886	103794214
Paired Reads (QC-failed)	0	0
Read1	64146443	51897107
Read1 (QC-failed)	0	0
Read2	64146443	51897107
Read2 (QC-failed)	0	0
Properly Paired Reads	120432240	96600514
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	93.89999999999999	93.10000000000001
With itself	121068112	97158250
With itself (QC-failed)	0	0
Singletons	1064424	862829
Singletons (QC-failed)	0	0
% Singleton	0.8	0.8
Diff. Chroms	130375	104207
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	53210709	43087725
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	9365263	7663784
Paired Optical Duplicate Reads	166423	111101
% Duplicate Reads	17.6003	17.7865

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	115903489	92448196
Rm = Number of Mitochondrial Reads	13561406	12224282
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.10474967750910391	0.11678601895715128

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	84975586	68280798
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	84975586	68280798
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	84975586	68280798
Paired Reads (QC-failed)	0	0
Read1	42487793	34140399
Read1 (QC-failed)	0	0
Read2	42487793	34140399
Read2 (QC-failed)	0	0
Properly Paired Reads	84975586	68280798
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	84975586	68280798
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	0.40598636320353493	0.4118596180712808
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.5195543442451107	1.524778974715211
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	0.560704141540136	0.5914717926993179
Fraction of Reads in blacklist regions	0.0024718158460242923	0.0024847395603080093
Fraction of Reads in promoter regions	0.19034670734721382	0.21680033675060448
Fraction of Reads in enhancer regions	0.37059047759906005	0.3755125855441818

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	47659434	38087846
Distinct Fragments	42516761	34167116
Positions with Two Read	3590660	2706676
NRF = Distinct/Total	0.892095	0.897061
PBC1 = OneRead/Distinct	0.903993	0.909989
PBC2 = OneRead/TwoRead	10.704123	11.487045

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	163679	95020
N1	142199	77659
N2	138186	74036
Np	167073	98522
N optimal	167073	98522
N conservative	163679	95020
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0207357083071134	1.0368553988633973
Self Consistency Ratio	1.0290405685091109	1.0489356529256038
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	187082	182600

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	236.0	236.0	561.0	351.0
50 percentile (median)	399.0	407.0	814.0	579.0
75 percentile	744.0	751.0	1079.0	910.0
Max size	3110.0	2566.0	2566.0	2566.0
Mean	525.6645535112945	529.9569112814896	843.0074501126652	665.5672430614163

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	25.351738670331297	27.616144704299405

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	0.19961013421631235	0.17846893524470384
Synthetic AUC	0.49489686404863925	0.49430282870739745
X-intercept	0.1329815419500227	0.14629469293571176
Synthetic X-intercept	0.0	2.3412220941098337e-264
Elbow Point	0.7641564705153098	0.7877994109260619
Synthetic Elbow Point	0.5006484047201136	0.5034496795955353
Synthetic JS Distance	0.43504543830754916	0.4671286064416142

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	0.32194438765035405	0.357786474610329	0.31475220859901554	0.3490887042916896	0.3148583244805943	0.3493181010953651	0.3513326009309994	0.3378508307268732	0.3380268932360271

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	0.3321735882793633	0.3041857457740862	0.33846947717277703	0.33387355661477697

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	0.2883373067186552	0.2577108441476355	0.28655814772404975	0.29203419676142167

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