QC Report

	general
Report generated at	2020-01-31 04:37:39
Title	Engrafment (yng)
Description	ATAC-seq Engrafment (yng)
Pipeline version	v1.6.0.1
Pipeline type	atac
Genome	mm10
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}, 'rep3': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	rep3
Total Reads	75384550	100913816	99443840
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	73762290	97355093	97005112
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	97.8	96.5	97.5
Paired Reads	75384550	100913816	99443840
Paired Reads (QC-failed)	0	0	0
Read1	37692275	50456908	49721920
Read1 (QC-failed)	0	0	0
Read2	37692275	50456908	49721920
Read2 (QC-failed)	0	0	0
Properly Paired Reads	72193162	94526490	94607560
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	95.8	93.7	95.1
With itself	72824146	95263904	95440786
With itself (QC-failed)	0	0	0
Singletons	938144	2091189	1564326
Singletons (QC-failed)	0	0	0
% Singleton	1.2	2.1	1.6
Diff. Chroms	53831	70126	70521
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	rep3
Unpaired Reads	0	0	0
Paired Reads	26364228	35674120	36543113
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	9063666	16578060	13481582
Paired Optical Duplicate Reads	760411	1485779	1249474
% Duplicate Reads	34.3787	46.470800000000004	36.8923

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	rep3
Rn = Number of Non-mitochondrial Reads	73553542	96595416	96587216
Rm = Number of Mitochondrial Reads	578851	2014439	1157083
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.007808340950224014	0.020428374020020616	0.011837856650851832

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	rep3
Total Reads	34364512	37622996	45714078
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	34364512	37622996	45714078
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	34364512	37622996	45714078
Paired Reads (QC-failed)	0	0	0
Read1	17182256	18811498	22857039
Read1 (QC-failed)	0	0	0
Read2	17182256	18811498	22857039
Read2 (QC-failed)	0	0	0
Properly Paired Reads	34364512	37622996	45714078
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	34364512	37622996	45714078
With itself (QC-failed)	0	0	0
Singletons	0	0	0
Singletons (QC-failed)	0	0	0
% Singleton	0.0	0.0	0.0
Diff. Chroms	0	0	0
Diff. Chroms (QC-failed)	0	0	0

Filtered and duplicates removed

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2	rep3
Fraction of reads in NFR	0.47297594987307257	0.3185831865409813	0.35030294130509704
Fraction of reads in NFR (QC pass)	True	False	False
Fraction of reads in NFR (QC reason)	OK	out of range [0.4, inf]	out of range [0.4, inf]
NFR / mono-nuc reads	1.690775673837563	1.0663566677635306	1.0921757100254188
NFR / mono-nuc reads (QC pass)	False	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True	True
Presence of Mono-Nuc peak	True	True	True
Presence of Di-Nuc peak	False	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	rep3
Total Fragments	26126347	34839343	36065741
Distinct Fragments	17188890	18821435	22865316
Positions with Two Read	3783493	4517375	5151083
NRF = Distinct/Total	0.657914	0.540235	0.63399
PBC1 = OneRead/Distinct	0.66343	0.550931	0.641452
PBC2 = OneRead/TwoRead	3.014049	2.295427	2.847363

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	rep3
Estimated library size by Picard tools	43541505.0	44741018.0	57671123.0

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	80333	30400
N1	24442	1051
N2	61592	36838
N3	33090	11670
Np	97919	40241
N optimal	97919	40241
N conservative	80333	30400
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep2_vs_rep3	rep2_vs_rep3
Rescue Ratio	1.2189137714264375	1.323717105263158
Self Consistency Ratio	2.5199247197447017	35.05042816365366
Reproducibility Test	borderline	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 raw peaks

	rep1	rep2	rep3
Number of peaks	181027	208643	299119

Top 300000 raw peaks from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	rep3	idr_opt	overlap_opt
Min size	73.0	73.0	73.0	73.0	73.0
25 percentile	73.0	82.0	82.0	658.0	276.0
50 percentile (median)	119.0	145.0	146.0	886.0	522.0
75 percentile	171.0	257.0	228.0	1121.0	863.0
Max size	3304.0	2448.0	3303.0	2697.0	2697.0
Mean	143.9944317698465	253.49088634653452	196.0902249606344	902.7222484530703	601.6399881534738

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (filtered BAM)

	rep1	rep2	rep3
Number of Subsampled Reads	25000000	25000000	25000000
Estimated Fragment Length	0	0	0
Cross-correlation at Estimated Fragment Length	0.18889831324049	0.178813793676478	0.204186010224665
Phantom Peak	145	145	150
Cross-correlation at Phantom Peak	0.1670618	0.1538705	0.1816818
Argmin of Cross-correlation	1500	1500	1500
Minimum of Cross-correlation	0.164304	0.1496019	0.1784186
NSC (Normalized Strand Cross-correlation coeff.)	1.149688	1.195264	1.144421
RSC (Relative Strand Cross-correlation coeff.)	8.918188	6.843544	7.8964

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	rep3
TSS enrichment	3.2073622172949197	6.788052930603195	4.478746477230261

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	rep3
AUC	0.2759840167704477	0.2757135283996076	0.2828492621075567
Synthetic AUC	0.4948319237507945	0.4952581080002051	0.49567017925184886
X-intercept	0.1614911179185296	0.14465475457301222	0.14371895670535165
Synthetic X-intercept	0.0	0.0	0.0
Elbow Point	0.5411311156790133	0.5527566045734121	0.5374499108192631
Synthetic Elbow Point	0.5015983853336383	0.5003454058976142	0.503880209595529
Synthetic JS Distance	0.2544993777234141	0.2688620593213536	0.25695992049397

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep3	rep1-pr1	rep2-pr1	rep3-pr1	rep1-pr2	rep2-pr2	rep3-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.07066391630994207	0.0989382929525336	0.11080400659070494	0.13084341194776752	0.14858976143207733	0.08125269063710787	0.13810514754290706	0.14347873837585928	0.08075871423060153	0.09226278395263085	0.1007691586964825	0.10820800848355618

FRiP for overlap peaks

	rep1_vs_rep2	rep1_vs_rep3	rep2_vs_rep3	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.025100766271747604	0.02616669923207322	0.034293794477841616	0.01494597100636843	0.04814818575320264	0.023470319143262607	0.04012158340840029

FRiP for IDR peaks

	rep1_vs_rep2	rep1_vs_rep3	rep2_vs_rep3	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.00477057293008779	0.005430784934367834	0.016399056848732694	0.002205298303086626	0.0341033978261593	0.010313431236653181	0.020290729132570906

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	rep3
Fraction of Reads in universal DHS regions	0.393790809542123	0.4241838688232059	0.41445188941577255
Fraction of Reads in blacklist regions	0.0026860267941532243	0.004012094092666092	0.002781200137078123
Fraction of Reads in promoter regions	0.0274493058420268	0.052011142334331906	0.03473118280981189
Fraction of Reads in enhancer regions	0.36283719087877636	0.36734325995728784	0.3760714806497902

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

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.