QC Report

	general
Report generated at	2020-04-15 15:44:42
Title	ENCSR356KRQ (subsampled 1/400)
Description	ATAC-seq on primary keratinocytes in day 0.0 of differentiation
Pipeline version	v1.7.1
Pipeline type	atac
Genome	hg38
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	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	249183	301770
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	960	1855
Paired Optical Duplicate Reads	3	6
% Duplicate Reads	0.38530000000000003	0.6147

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	461170	550916
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	461170	550916
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	461170	550916
Paired Reads (QC-failed)	0	0
Read1	230585	275458
Read1 (QC-failed)	0	0
Read2	230585	275458
Read2 (QC-failed)	0	0
Properly Paired Reads	461170	550916
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	461170	550916
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	0.5035996900871412	0.5476324967292632
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.5384349444850742	1.815339639280494
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	230808	275697
Distinct Fragments	230603	275503
Positions with Two Read	197	175
NRF = Distinct/Total	0.999112	0.999296
PBC1 = OneRead/Distinct	0.999133	0.999339
PBC2 = OneRead/TwoRead	1169.558376	1573.262857

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	30041	374
N1	13440	45
N2	14918	75
Np	30250	478
N optimal	30250	478
N conservative	30041	374
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0069571585499817	1.2780748663101604
Self Consistency Ratio	1.109970238095238	1.6666666666666667
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	236483	270505

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	168.0	150.0
25 percentile	150.0	150.0	373.25	229.0
50 percentile (median)	182.0	190.0	461.0	311.0
75 percentile	249.0	255.0	559.5	411.0
Max size	976.0	1058.0	907.0	1212.0
Mean	209.35772127383365	214.82435075137244	471.8661087866109	332.85738842975206

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (filtered BAM)

	rep1	rep2
Number of Subsampled Reads	230808	275697
Estimated Fragment Length	0	0
Cross-correlation at Estimated Fragment Length	0.0111972879681259	0.0135895835739089
Phantom Peak	70	70
Cross-correlation at Phantom Peak	0.01143303	0.01393697
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.004724189	0.007344452
NSC (Normalized Strand Cross-correlation coeff.)	2.370203	1.85032
RSC (Relative Strand Cross-correlation coeff.)	0.9648616	0.9473054

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

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	rep2
TSS enrichment	18.987184149250922	16.735814540085777

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	0.014306630744699232	0.01693557563490223
Synthetic AUC	0.4367909846533249	0.44145999871653496
X-intercept	0.9579879480012636	0.9507323568575233
Synthetic X-intercept	0.03690139395640774	0.018356871833377548
Elbow Point	0.958491780728798	0.9513541504220598
Synthetic Elbow Point	0.5967858194052946	0.45630251527123744
Synthetic JS Distance	0.09990741508026403	0.09641430432679374

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	0.9243489385692911	0.918188979808174	0.970522928538593	0.9672835786217863	0.9708739548277417	0.9674650944971647	0.6989415919200542	0.9209772272766795	0.9224945755490651

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	0.1939825271765443	0.15084025413621874	0.14622737404613406	0.19479075888807867

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	0.0126540629946467	0.004753127913784505	0.007077303981006179	0.014580776732412067

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	0.46376173645293495	0.4277004116780054
Fraction of Reads in blacklist regions	0.0006613613201205629	0.0007532908828206115
Fraction of Reads in promoter regions	0.15181603313311795	0.13602799700861837
Fraction of Reads in enhancer regions	0.3902769043953423	0.372750110724684

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.