QC Report

	general
Report generated at	2021-08-31 21:28:35
Title	set-16_RW12223_L4larva_1_3
Description	gevirl
Pipeline version	v1.3.6
Pipeline type	tf
Genome	WS245chr
Aligner	bwa
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}, 'ctl1': {'paired_end': True}}
Peak caller	spp

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	ctl1
Total Reads	17790760	27163804	19611464
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	13316225	21486037	18793459
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	74.8	79.10000000000001	95.8
Paired Reads	17790760	27163804	19611464
Paired Reads (QC-failed)	0	0	0
Read1	8895380	13581902	9805732
Read1 (QC-failed)	0	0	0
Read2	8895380	13581902	9805732
Read2 (QC-failed)	0	0	0
Properly Paired Reads	12509390	20370684	18604258
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	70.3	75.0	94.89999999999999
With itself	13267434	21415738	18775408
With itself (QC-failed)	0	0	0
Singletons	48791	70299	18051
Singletons (QC-failed)	0	0	0
% Singleton	0.3	0.3	0.1
Diff. Chroms	114433	154334	85162
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	5246988	8789689	8448102
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	2779225	4280772	701192
Paired Optical Duplicate Reads	26095	41882	36175
% Duplicate Reads	52.968	48.7022	8.3

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	ctl1
Total Reads	4935526	9017834	15493820
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	4935526	9017834	15493820
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	4935526	9017834	15493820
Paired Reads (QC-failed)	0	0	0
Read1	2467763	4508917	7746910
Read1 (QC-failed)	0	0	0
Read2	2467763	4508917	7746910
Read2 (QC-failed)	0	0	0
Properly Paired Reads	4935526	9017834	15493820
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	4935526	9017834	15493820
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

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	ctl1
Total Fragments	5207706	8721549	8322847
Distinct Fragments	2462060	4495541	7637433
Positions with Two Read	679969	1249915	592898
NRF = Distinct/Total	0.472772	0.515452	0.917647
PBC1 = OneRead/Distinct	0.424681	0.474944	0.916588
PBC2 = OneRead/TwoRead	1.537702	1.708219	11.807053

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. See more details at the ENCODE portal standard for ChIP-Seq pipeline

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	24752	5795
N1	20843	5876
N2	29107	4377
Np	28050	6905
N optimal	28050	6905
N conservative	24752	5795
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.1332417582417582	1.1915444348576358
Self Consistency Ratio	1.3964880295542867	1.3424720127941512
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	36624	44899

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	119.0	106.0	111.0	111.0
25 percentile	476.0	424.0	276.0	444.0
50 percentile (median)	476.0	424.0	444.0	444.0
75 percentile	476.0	424.0	444.0	444.0
Max size	2265.0	2105.0	2209.0	2209.0
Mean	467.8236402359109	418.33726809060335	396.08269370021725	431.20288770053475

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (trimmed/filtered SE BAM)

	rep1	rep2
Number of Subsampled Reads	5022727	8469430
Estimated Fragment Length	230	205
Cross-correlation at Estimated Fragment Length	0.564238026842004	0.638652716155753
Phantom Peak	55	55
Cross-correlation at Phantom Peak	0.4436954	0.6050273
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.3720568	0.5812142
NSC (Normalized Strand Cross-correlation coeff.)	1.516538	1.098825
RSC (Relative Strand Cross-correlation coeff.)	2.682647	2.412059

Performed on subsampled (15000000) reads mapped from FASTQs that are trimmed to 50. Such FASTQ trimming and subsampling reads are for cross-corrleation analysis only. Untrimmed FASTQs are used for all the other analyses.

NOTE1: For SE datasets, reads from replicates are randomly subsampled to 15000000.
NOTE2: For PE datasets, the first end (R1) of each read-pair is selected and trimmed to 50 the reads are then randomly subsampled to 15000000.

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

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.25159094225296336	0.32023525270358416
Synthetic AUC	0.49709219074025934	0.4978525846975484
X-intercept	0.02100739699888626	0.01934876071327348
Synthetic X-intercept	0.0	0.0
Elbow Point	0.796899887029105	0.6893340305859716
Synthetic Elbow Point	0.5012552694129042	0.5030225185916034
JS Distance	0.3357601491879693	0.1961892460389527
Synthetic JS Distance	0.39672543325481135	0.2721969862349074
% Genome Enriched	17.746210684731366	28.126758932884112
Diff. Enrichment	32.46174014703647	21.265316474280866
CHANCE Divergence	0.2890627231939256	0.18335018956062457

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for spp raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.4959929296289798	0.39917623234138044	0.5322072937282495	0.43221389255692827	0.5323224038622849	0.43206726406080753	0.4162533611975897	0.4322127624564227	0.43345428870301883

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	0.32548869949603537	0.4112617378573226	0.31864702765653036	0.3483873418302115

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	0.18112504801710844	0.2893782749802149	0.11017423917982966	0.19728918339382057

For spp 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