QC Report

	general
Report generated at	2019-11-16 00:05:16
Title	ENCSR356KRQ (subsampled 1/400)
Description	ATAC-seq on primary keratinocytes in day 0.0 of differentiation
Pipeline version	v1.5.3
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	OrderedDict([('rep1', {'paired_end': True}), ('rep2', {'paired_end': True})])
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	691166	848854
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	680479	838515
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.5	98.8
Paired Reads	691166	848854
Paired Reads (QC-failed)	0	0
Read1	345583	424427
Read1 (QC-failed)	0	0
Read2	345583	424427
Read2 (QC-failed)	0	0
Properly Paired Reads	556054	682808
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	80.5	80.4
With itself	676210	832920
With itself (QC-failed)	0	0
Singletons	4269	5595
Singletons (QC-failed)	0	0
% Singleton	0.6	0.7000000000000001
Diff. Chroms	15177	20376
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	245210	296392
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	882	1676
Paired Optical Duplicate Reads	2	6
% Duplicate Reads	0.3597	0.5655

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	665674	817862
Rm = Number of Mitochondrial Reads	54279	77218
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.07539242144973353	0.08626938374223533

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	456690	545132
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	456690	545132
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	456690	545132
Paired Reads (QC-failed)	0	0
Read1	228345	272566
Read1 (QC-failed)	0	0
Read2	228345	272566
Read2 (QC-failed)	0	0
Properly Paired Reads	456690	545132
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	456690	545132
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	0.5025463237588045	0.5463799114259104
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.5317476261266691	1.805493716774327
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.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	228557	272785
Distinct Fragments	228358	272602
Positions with Two Read	191	169
NRF = Distinct/Total	0.999129	0.999329
PBC1 = OneRead/Distinct	0.99915	0.999362
PBC2 = OneRead/TwoRead	1194.575916	1612.0

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	30208	261
N1	14753	26
N2	15865	35
Np	32270	301
N optimal	32270	301
N conservative	30208	261
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.068260063559322	1.1532567049808429
Self Consistency Ratio	1.0753745001016743	1.3461538461538463
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	297936	299650

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	73.0	73.0	73.0	73.0
25 percentile	73.0	73.0	273.0	140.0
50 percentile (median)	73.0	73.0	359.0	204.0
75 percentile	116.0	129.0	426.0	283.0
Max size	860.0	1108.0	759.0	1208.0
Mean	100.73481888727781	106.7660270315368	359.40199335548175	225.2372482181593

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	19.06062750181121	16.757705401589984

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	0.014255843988670825	0.01687618984821658
Synthetic AUC	0.436685561986168	0.4413355590403154
X-intercept	0.9581259022004694	0.9509082984449163
Synthetic X-intercept	0.03741526373241471	0.018746630087640406
Elbow Point	0.9586297349280039	0.9515280926732325
Synthetic Elbow Point	0.59968520973226	0.46029700985413047
Synthetic JS Distance	0.10013462212316575	0.09569441467036811

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	0.9893866736736079	0.8944475833376136	0.9966104070139175	0.996180741545167	0.9962381319412815	0.9958468774535342	0.6275605846148318	0.9538202318970198	0.9613703060430018

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	0.18011183623438096	0.14732312947513632	0.1404063602943874	0.18667986927817518

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	0.008217028573938285	0.0029779500317502023	0.0042833662305643404	0.00857537566553739

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

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.46616961177166133	0.43002061885928544
Fraction of Reads in blacklist regions	0.0005758829840811053	0.000680569109866968
Fraction of Reads in promoter regions	0.15275788828308043	0.13694297894821805
Fraction of Reads in enhancer regions	0.39283540257067157	0.3754631905666884

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.