QC Report

	general
Report generated at	2019-07-26 14:04:18
Title	ENCSR356KRQ (subsampled 1/400 reads)
Description	ATAC-seq on primary keratinocytes in day 0.0 of differentiation
Pipeline version	v1.4.2
Pipeline type	atac
Genome	hg38_chr19_chrM.tsv
Paired-end per replicate	[True, True]
Aligner	bowtie2
Peak caller	macs2
Control paired-end per replicate	[True, True]

Alignment quality metrics

SAMstat (raw BAM)

	rep1	rep2
Total	691166	848854
Total(QC-failed)	0	0
Dupes	0	0
Dupes(QC-failed)	0	0
Mapped	119582	163657
Mapped(QC-failed)	0	0
% Mapped	0.0	0.0
Paired	691166	848854
Paired(QC-failed)	0	0
Read1	345583	424427
Read1(QC-failed)	0	0
Read2	345583	424427
Read2(QC-failed)	0	0
Properly Paired	87396	121978
Properly Paired(QC-failed)	0	0
% Properly Paired	0.0	0.0
With itself	110874	153552
With itself(QC-failed)	0	0
Singletons	8708	10105
Singletons(QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	6	8
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAMs)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	30210	41934
Unmapped Reads	0	0
Unpaired Dupes	0	0
Paired Dupes	956	2051
Paired Opt. Dupes	0	1
% Dupes/100	0.031645	0.04891

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

	rep1	rep2
Rn = Number of Mitochondrial Reads	636718	771370
Rm = Number of Non-mitochondrial Reads	691166	848854
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.520501790819	0.523911508532

Preseq performs a yield prediction by subsampling the reads, calculating the number of distinct reads, and then extrapolating out to see where the expected number of distinct reads no longer increases. The confidence interval gives a gauge as to the validity of the yield predictions.

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total	58508	79766
Total(QC-failed)	0	0
Dupes	0	0
Dupes(QC-failed)	0	0
Mapped	58508	79766
Mapped(QC-failed)	0	0
% Mapped	100.0	100.0
Paired	58508	79766
Paired(QC-failed)	0	0
Read1	29254	39883
Read1(QC-failed)	0	0
Read2	29254	39883
Read2(QC-failed)	0	0
Properly Paired	58508	79766
Properly Paired(QC-failed)	0	0
% Properly Paired	100.0	100.0
With itself	58508	79766
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

	rep1	rep2
Fraction of reads in NFR	0.697251217815	0.729131045043
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	3.29155716163	3.77174917492
NFR / mono-nuc reads (QC pass)	True	True
NFR / mono-nuc reads (QC reason)	OK	OK
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	False	False
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.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Reads (Pairs)	9186	11856
Distinct Reads (Pairs)	9176	11846
One Read (Pair)	9168	11839
Two Reads (Pairs)	7	6
NRF = Distinct/Total	0.998911	0.999157
PBC1 = OnePair/Distinct	0.999128	0.999409
PBC2 = OnePair/TwoPair	1309.714286	1973.166667

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

	rep1	rep2
Estimated library size by Picard tools	51184121.0	41551204.0

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	1791	42
N1	610	1
N2	842	7
Np	1820	43
N optimal	1820	43
N conservative	1791	42
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.01619207147	1.02380952381
Self Consistency Ratio	1.38032786885	7.0
Reproducibility Test	pass	borderline

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 peaks called

	rep1	rep2	idr_opt	overlap_opt
Number of peaks	10815	13058	43	1820

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	73.0	73.0	85.0	73.0
25 percentile	73.0	73.0	208.0	133.0
50 percentile (median)	73.0	73.0	343.0	191.0
75 percentile	131.0	141.0	508.0	272.25
Max size	695.0	800.0	748.0	853.0
Mean	108.137216828	113.659901976	369.255813953	216.318131868

Alignment enrichment

Strand cross-correlation measures

	rep1	rep2
Reads	9176	11846
Est. Fragment Len.	0	0
Corr. Est. Fragment Len.	0.00941645271926	0.0122197346852
Phantom Peak	75	70
Corr. Phantom Peak	0.01134414	0.01387251
Argmin. Corr.	1500	1500
Min. Corr.	0.007060396	0.008253066
NSC	1.3337	1.48063
RSC	0.55	0.7058824

Performed on subsampled reads

NOTE1: For SE datasets, reads from replicates are randomly subsampled.
NOTE2: For PE datasets, the first end of each read-pair is selected and the reads are then randomly subsampled.

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

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	0.966107236269	0.965262535877	0.986486486486	0.982188080365	0.988448125545	0.983454330576	0.921415659785	0.96303872134	0.969270288269

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	0.21905622681	0.145215780296	0.16748269458	0.220887641518

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	0.0196223004472	0.000762859633827	0.00569812594969	0.0193844543811

For raw peaks (e.g. spp and macs2):

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

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.310265911072	0.291153131859
Fraction of Reads in blacklist regions	0.0	0.0
Fraction of Reads in promoter regions	0.157367044464	0.150050650008
Fraction of Reads in enhancer regions	0.206244551003	0.205512409252

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.

Other quality metrics

Sequence quality metrics (GC bias)

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

Comparison to Roadmap DNase

This bar chart shows the correlation between the Roadmap DNase samples to your sample, when the signal in the universal DNase peak region sets are compared. The closer the sample is in signal distribution in the regions to your sample, the higher the correlation.