QC Report

	general
Report generated at	2020-02-05 10:24:57
Title	Engrafment (old)
Description	ATAC-seq Engrafment (old)
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	103191668	117623518	113431192
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	100751744	114630557	110603504
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	97.6	97.5	97.5
Paired Reads	103191668	117623518	113431192
Paired Reads (QC-failed)	0	0	0
Read1	51595834	58811759	56715596
Read1 (QC-failed)	0	0	0
Read2	51595834	58811759	56715596
Read2 (QC-failed)	0	0	0
Properly Paired Reads	98450146	112091028	107884334
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	95.39999999999999	95.3	95.1
With itself	99273578	113091628	108733744
With itself (QC-failed)	0	0	0
Singletons	1478166	1538929	1869760
Singletons (QC-failed)	0	0	0
% Singleton	1.4000000000000001	1.3	1.6
Diff. Chroms	71299	85405	77901
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	rep3
Unpaired Reads	0	0	0
Paired Reads	36281880	42786949	39097409
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	14490765	18803833	16568740
Paired Optical Duplicate Reads	1195821	1490862	1408158
% Duplicate Reads	39.939400000000006	43.947599999999994	42.3781

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	100405134	114020894	110167974
Rm = Number of Mitochondrial Reads	926718	1686381	1189806
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.00914537711202594	0.01457454598252357	0.01068453412056167

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	43233094	47471984	44663354
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	43233094	47471984	44663354
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	43233094	47471984	44663354
Paired Reads (QC-failed)	0	0	0
Read1	21616547	23735992	22331677
Read1 (QC-failed)	0	0	0
Read2	21616547	23735992	22331677
Read2 (QC-failed)	0	0	0
Properly Paired Reads	43233094	47471984	44663354
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	43233094	47471984	44663354
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	0.4201124188445341	0.40703579997780504	0.33920748821447316
Fraction of reads in NFR (QC pass)	True	True	False
Fraction of reads in NFR (QC reason)	OK	OK	out of range [0.4, inf]
NFR / mono-nuc reads	1.4133775603956116	1.275601178690659	1.0741825974573367
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	False

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	35896109	42088536	38604077
Distinct Fragments	21624096	23749070	22339148
Positions with Two Read	4974174	5615727	5223428
NRF = Distinct/Total	0.602408	0.564265	0.578673
PBC1 = OneRead/Distinct	0.613597	0.576371	0.591068
PBC2 = OneRead/TwoRead	2.667473	2.43749	2.527832

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	51306387.0	52261294.0	52860515.0

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	42349	3734
N1	5856	412
N2	55689	33284
N3	10074	612
Np	70058	29570
N optimal	70058	29570
N conservative	42349	3734
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep2_vs_rep3	rep2_vs_rep3
Rescue Ratio	1.6543011641361072	7.919121585431173
Self Consistency Ratio	9.509733606557377	80.7864077669903
Reproducibility Test	borderline	fail

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	288126	186525	299145

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	98.0	73.0	521.0	227.0
50 percentile (median)	107.0	161.0	118.0	731.0	413.0
75 percentile	150.0	336.0	174.0	952.0	714.0
Max size	1644.0	2921.0	3044.0	2768.0	2768.0
Mean	123.97736754058988	299.51371665996516	142.03646726503868	749.1535339871491	501.4484855405521

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	0.191316493579889	0.196109422830772	0.191027316759835
Phantom Peak	145	145	145
Cross-correlation at Phantom Peak	0.1710109	0.1741907	0.1681984
Argmin of Cross-correlation	1500	1500	1500
Minimum of Cross-correlation	0.1687805	0.1684871	0.1659469
NSC (Normalized Strand Cross-correlation coeff.)	1.133522	1.163943	1.151135
RSC (Relative Strand Cross-correlation coeff.)	10.10419	4.842958	11.13934

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	2.151712769672642	7.673244587898115	2.645575351127997

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	0.2939009061104606	0.2786092403384647	0.29447058560694483
Synthetic AUC	0.4954328533324549	0.4956766938789375	0.49562498909435554
X-intercept	0.14184049717658728	0.14519180010557803	0.13796530321693087
Synthetic X-intercept	0.0	0.0	0.0
Elbow Point	0.5195179402684241	0.5451446099211364	0.5216874890022876
Synthetic Elbow Point	0.4988443358215734	0.5024962976934453	0.5026287760685217
Synthetic JS Distance	0.23756391970389742	0.26616864400227414	0.23925791633341062

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	0.08558545451315605	0.08867984114588512	0.0945070314244649	0.05663790536768405	0.0954512455177774	0.05253877473963219	0.05329061358831332	0.09778533797955442	0.06269260757678913	0.07690329899071299	0.08749009998165304	0.08114197795072275

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	0.01220396052160817	0.012022130831802794	0.016718528585748856	0.0035597035918826445	0.044300549983333326	0.007362904272706434	0.025427996388404647

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	0.0004898557146617462	0.0008711410648532887	0.0026659612929549187	0.0006166109693652738	0.03049404886890761	0.0020084698520402206	0.013349988422707001

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	0.38575055488742027	0.42670542271837636	0.3827142045803367
Fraction of Reads in blacklist regions	0.0026358973984142798	0.003536043490408996	0.0026451215464024487
Fraction of Reads in promoter regions	0.0216920861597368	0.05128188870302956	0.023350037706527816
Fraction of Reads in enhancer regions	0.3606073856291664	0.371091568450141	0.3559101942948575

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.