QC Report

	general
Report generated at	2019-07-13 16:41:39
Title	ENCSR889WQX
Description	ATAC-seq on Mus musculus C57BL/6 frontal cortex adult
Pipeline version	v1.4.2
Genome	mm10_google.tsv
Paired-end per replicate	[False, False]
Aligner	bowtie2
Peak caller	macs2
Control paired-end per replicate	[False, False]

Alignment quality metrics

SAMstat (raw BAM)

	rep1	rep2
Total	79937574	67703560
Total(QC-failed)	0	0
Dupes	0	0
Dupes(QC-failed)	0	0
Mapped	67418828	65975170
Mapped(QC-failed)	0	0
% Mapped	0.0	0.0
Paired	0	0
Paired(QC-failed)	0	0
Read1	0	0
Read1(QC-failed)	0	0
Read2	0	0
Read2(QC-failed)	0	0
Properly Paired	0	0
Properly Paired(QC-failed)	0	0
% Properly Paired	0.0	0.0
With itself	0	0
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

Marking duplicates (filtered BAMs)

	rep1	rep2
Unpaired Reads	60738378	58655573
Paired Reads	0	0
Unmapped Reads	0	0
Unpaired Dupes	27075809	15938588
Paired Dupes	0	0
Paired Opt. Dupes	0	0
% Dupes/100	0.445778	0.271732

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
Total reads	110773830	102194989
Mito. reads	23249263	8122611
Frac. of mito. reads	0.209880465449	0.0794814998219
Total deduped reads	33662569	42716985
Mito. deduped reads	31192	29699
Frac. of deduped mito. reads	0.000926607829604	0.000695250378743
Total dup reads	27075809	15938588
Mito. dup reads	18235849	6296240
Frac. of mito. dup reads	0.673510771183	0.395031228613

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	33662569	42716985
Total(QC-failed)	0	0
Dupes	0	0
Dupes(QC-failed)	0	0
Mapped	33662569	42716985
Mapped(QC-failed)	0	0
% Mapped	100.0	100.0
Paired	0	0
Paired(QC-failed)	0	0
Read1	0	0
Read1(QC-failed)	0	0
Read2	0	0
Read2(QC-failed)	0	0
Properly Paired	0	0
Properly Paired(QC-failed)	0	0
% Properly Paired	0.0	0.0
With itself	0	0
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

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Reads (Pairs)	42471337	52329634
Distinct Reads (Pairs)	36049091	44133394
One Read (Pair)	33945290	39589215
Two Reads (Pairs)	1695966	3436845
NRF = Distinct/Total	0.848786	0.843373
PBC1 = OnePair/Distinct	0.941641	0.897035
PBC2 = OnePair/TwoPair	20.015313	11.519057

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	251111	125042
N1	214343	107876
N2	231203	107745
Np	253649	135148
N optimal	253649	135148
N conservative	251111	125042
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.01010708412	1.0808208442
Self Consistency Ratio	1.07865897183	1.00121583368
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 peaks called

	rep1	rep2	idr_opt	overlap_opt
Number of peaks	299596	299568	135148	253649

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	73.0	73.0	73.0	73.0
25 percentile	125.0	168.0	423.0	262.0
50 percentile (median)	225.0	287.0	626.0	433.0
75 percentile	424.0	509.0	892.0	695.0
Max size	2059.0	2177.0	2447.0	2447.0
Mean	315.005217026	377.243814426	684.464350194	520.343963509

Alignment enrichment

Strand cross-correlation measures

	rep1	rep2
Reads	25000000	25000000
Est. Fragment Len.	0	0
Corr. Est. Fragment Len.	0.364692997939	0.36894494402
Phantom Peak	45	45
Corr. Phantom Peak	0.3369163	0.3397161
Argmin. Corr.	1500	1500
Min. Corr.	0.2830327	0.2878876
NSC	1.288519	1.281559
RSC	1.515494	1.563952

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

TSS enrichment

	rep1	rep2
TSS enrichment	12.0910218326	9.83151375332

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/

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	0.250243396219	0.25948670056	0.25526316525	0.253116115182	0.252431479461	0.252864424316	0.260806626028	0.25645427965	0.254138443884

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	0.240096370661	0.215947179326	0.231784775448	0.24060551218

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	0.157061215813	0.143912840679	0.14158452238	0.165157990255

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	0.526166502192	0.547866219464
Fraction of Reads in blacklist regions	0.000417764636875	0.000360435189063
Fraction of Reads in promoter regions	0.0991146154973	0.114257252148
Fraction of Reads in enhancer regions	0.427153726117	0.434060647472

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.