QC Report

	general
Report generated at	2019-12-27 06:46:09
Title	ENCODE_GM12878_DNase-seq
Description	ENCODE GM12878 DNase-seq Samples from Stam and Crawford labs
Pipeline version	v1.5.4
Pipeline type	dnase
Genome	hg38
Aligner	bowtie2
Sequencing endedness	OrderedDict([('rep1', {'paired_end': False}), ('rep2', {'paired_end': False}), ('rep3', {'paired_end': False}), ('rep4', {'paired_end': False}), ('rep5', {'paired_end': False})])
Peak caller	macs2

Alignment quality metrics

Marking duplicates (filtered BAM)

	rep1	rep2	rep3	rep4	rep5
Unpaired Reads	46947124	32398606	41234629	22900695	45012772
Paired Reads	0	0	0	0	0
Unmapped Reads	0	0	0	0	0
Unpaired Duplicate Reads	23546263	12941233	12318727	4670359	17664285
Paired Duplicate Reads	0	0	0	0	0
Paired Optical Duplicate Reads	0	0	0	0	0
% Duplicate Reads	50.154900000000005	39.9438	29.874699999999997	20.394000000000002	39.2428

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)

SAMstat (filtered/deduped BAM)

	rep1	rep2	rep3	rep4	rep5
Total Reads	23398320	19454653	28887133	18216429	27334517
Total Reads (QC-failed)	0	0	0	0	0
Duplicate Reads	0	0	0	0	0
Duplicate Reads (QC-failed)	0	0	0	0	0
Mapped Reads	23398320	19454653	28887133	18216429	27334517
Mapped Reads (QC-failed)	0	0	0	0	0
% Mapped Reads	100.0	100.0	100.0	100.0	100.0
Paired Reads	0	0	0	0	0
Paired Reads (QC-failed)	0	0	0	0	0
Read1	0	0	0	0	0
Read1 (QC-failed)	0	0	0	0	0
Read2	0	0	0	0	0
Read2 (QC-failed)	0	0	0	0	0
Properly Paired Reads	0	0	0	0	0
Properly Paired Reads (QC-failed)	0	0	0	0	0
% Properly Paired Reads	0.0	0.0	0.0	0.0	0.0
With itself	0	0	0	0	0
With itself (QC-failed)	0	0	0	0	0
Singletons	0	0	0	0	0
Singletons (QC-failed)	0	0	0	0	0
% Singleton	0.0	0.0	0.0	0.0	0.0
Diff. Chroms	0	0	0	0	0
Diff. Chroms (QC-failed)	0	0	0	0	0

Filtered and duplicates removed

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	rep4	rep5
Total Fragments	46926703	32384954	41201896	20732370	43644272
Distinct Fragments	23398740	19454957	28912911	18223701	27833424
Positions with Two Read	6263172	4589404	5141848	1017423	1220903
NRF = Distinct/Total	0.498623	0.600741	0.701737	0.878997	0.637734
PBC1 = OneRead/Distinct	0.4758	0.606721	0.730876	0.921029	0.92685
PBC2 = OneRead/TwoRead	1.777552	2.571954	4.109756	16.497133	21.129777

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	132200	84052
N1	90159	49899
N2	79611	44224
N3	108083	66353
N4	74543	44562
N5	107407	64798
Np	178815	116780
N optimal	178815	116780
N conservative	132200	84052
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep3	rep1_vs_rep3
Rescue Ratio	1.3526096822995461	1.3893780040927046
Self Consistency Ratio	1.4499416444199993	1.5003844066570189
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	rep3	rep4	rep5
Number of peaks	173207	178292	229990	134503	169490

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

Peak calling statistics

Peak region size

	rep1	rep2	rep3	rep4	rep5	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0	150.0	150.0	150.0
25 percentile	165.0	160.0	161.0	179.0	193.0	420.0	313.0
50 percentile (median)	227.0	218.0	214.0	268.0	320.0	631.0	481.0
75 percentile	379.0	362.0	354.0	479.0	532.0	966.0	783.0
Max size	2235.0	1943.0	1887.0	2625.0	2264.0	2628.0	2628.0
Mean	322.2292286108529	314.2238182307675	319.15976346797686	399.9251838248961	404.49299663696974	726.6419078609351	600.164700947907

Enrichment / Signal-to-noise ratio

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2	rep3	rep4	rep5
AUC	0.1745289578226782	0.15372805075119064	0.19414889103761335	0.17450260470926068	0.19522475842165743
Synthetic AUC	0.4837631871063979	0.482185983489757	0.48540097282961825	0.48629941736923804	0.48872546373792825
X-intercept	0.2758578634735998	0.3270487657122521	0.23097285506640783	0.25224574991952686	0.18570397487239262
Synthetic X-intercept	1.1307209743140507e-31	3.325813669124358e-26	2.378065482276183e-39	8.699808992158127e-45	1.4369727296302043e-66
Elbow Point	0.6699902232654293	0.7388952090001939	0.6555990299240054	0.6748565978963026	0.7116782994479843
Synthetic Elbow Point	0.5032631336020629	0.49221193287651444	0.5075883908951274	0.5208825733106693	0.4999432588881882
Synthetic JS Distance	0.3669404660131944	0.38464066094279564	0.3559361888813847	0.40298217600084585	0.3965298526132157

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep3	rep4	rep5	rep1-pr1	rep2-pr1	rep3-pr1	rep4-pr1	rep5-pr1	rep1-pr2	rep2-pr2	rep3-pr2	rep4-pr2	rep5-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.20086809651291204	0.22330334033714197	0.21934584661006157	0.25619433973585054	0.2645960782844636	0.1977617196448292	0.20339421096874816	0.2170819170010305	0.23922294324409338	0.26526686879936934	0.19752913884415632	0.2034930257297843	0.2168590346788153	0.23915643615751672	0.2656334577133175	0.2660302614893137	0.24676003383680525	0.24657389969388158

FRiP for overlap peaks

	rep1_vs_rep2	rep1_vs_rep3	rep1_vs_rep4	rep1_vs_rep5	rep2_vs_rep3	rep2_vs_rep4	rep2_vs_rep5	rep3_vs_rep4	rep3_vs_rep5	rep4_vs_rep5	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	rep4-pr1_vs_rep4-pr2	rep5-pr1_vs_rep5-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.20626884967142217	0.2096867881754308	0.19360591766324325	0.19851236536399539	0.2088238541504773	0.19287816549752698	0.19718064632544655	0.19691030817892358	0.20267248713831	0.19697797156240454	0.16030945811494157	0.17335446692367115	0.17387422118296555	0.2223767347595953	0.23695432408774592	0.23564354632947582

FRiP for IDR peaks

	rep1_vs_rep2	rep1_vs_rep3	rep1_vs_rep4	rep1_vs_rep5	rep2_vs_rep3	rep2_vs_rep4	rep2_vs_rep5	rep3_vs_rep4	rep3_vs_rep5	rep4_vs_rep5	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	rep3-pr1_vs_rep3-pr2	rep4-pr1_vs_rep4-pr2	rep5-pr1_vs_rep5-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.1665352538289373	0.1741669043934718	0.1290346945490496	0.11631307491234127	0.1725251851117337	0.13341952558455414	0.11697779221778519	0.14451043040262262	0.14962744607542444	0.12845381914835735	0.12332509342551089	0.1331129884454891	0.14437655518699807	0.18122004043712409	0.19873063789640036	0.2066103801307233

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	rep4	rep5
Fraction of Reads in universal DHS regions	0.33854114312480554	0.3627000697468107	0.3354782586318996	0.3885799461573945	0.35979779704905707
Fraction of Reads in blacklist regions	2.3933342222860446e-06	2.724284005476736e-06	3.3204070348633747e-06	6.450221390811559e-05	5.158313205241563e-05
Fraction of Reads in promoter regions	0.12834147921731132	0.14496059117579738	0.13315018982697843	0.15746181647347018	0.10064820241747824
Fraction of Reads in enhancer regions	0.30606641844371735	0.31803106434229383	0.2941816300002649	0.31016435767954303	0.326500775557878

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.