QC Report

	general
Report generated at	2021-08-30 17:56:27
Title	hsf-1_RW12254_youngadult_1_5
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	15889712	21939426	15663024
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	15838115	21856017	15635356
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	99.7	99.6	99.8
Paired Reads	15889712	21939426	15663024
Paired Reads (QC-failed)	0	0	0
Read1	7944856	10969713	7831512
Read1 (QC-failed)	0	0	0
Read2	7944856	10969713	7831512
Read2 (QC-failed)	0	0	0
Properly Paired Reads	15783902	21757654	15544642
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	99.3	99.2	99.2
With itself	15829822	21844928	15627862
With itself (QC-failed)	0	0	0
Singletons	8293	11089	7494
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.0
Diff. Chroms	6377	4401	37100
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	7005394	9649039	7146754
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	873785	1146193	1037752
Paired Optical Duplicate Reads	67404	62740	85695
% Duplicate Reads	12.472999999999999	11.8788	14.5206

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	12263218	17005692	12218004
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	12263218	17005692	12218004
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	12263218	17005692	12218004
Paired Reads (QC-failed)	0	0	0
Read1	6131609	8502846	6109002
Read1 (QC-failed)	0	0	0
Read2	6131609	8502846	6109002
Read2 (QC-failed)	0	0	0
Properly Paired Reads	12263218	17005692	12218004
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	12263218	17005692	12218004
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	6948067	9578237	7039376
Distinct Fragments	6085160	8453074	6023188
Positions with Two Read	697298	915725	793762
NRF = Distinct/Total	0.875806	0.882529	0.855642
PBC1 = OneRead/Distinct	0.872532	0.880011	0.850773
PBC2 = OneRead/TwoRead	7.614384	8.123398	6.455793

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	71244	2572
N1	29630	1217
N2	58728	1877
Np	74788	2935
N optimal	74788	2935
N conservative	71244	2572
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0497445398910785	1.1411353032659408
Self Consistency Ratio	1.9820452244346947	1.542317173377157
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	110633	121653

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	78.0	62.0	76.0	76.0
25 percentile	310.0	250.0	304.0	304.0
50 percentile (median)	310.0	250.0	304.0	304.0
75 percentile	310.0	250.0	304.0	304.0
Max size	1890.0	1163.0	1623.0	1623.0
Mean	309.853009499878	249.83662548395847	292.5161839863714	303.5425201904049

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	6470641	8902333
Estimated Fragment Length	160	135
Cross-correlation at Estimated Fragment Length	0.668670804849711	0.738066300623505
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.667683	0.7372111
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.6611888	0.7285628
NSC (Normalized Strand Cross-correlation coeff.)	1.011316	1.013044
RSC (Relative Strand Cross-correlation coeff.)	1.1521	1.09888

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	0.3992731910290915	0.3957568773315546
Synthetic AUC	0.49817215646314067	0.498416456852257
X-intercept	0.01935382115184584	0.01975111952292252
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5673680280624417	0.5686757186808097
Synthetic Elbow Point	0.4987520428590393	0.5007818341046567
JS Distance	0.08324693291572466	0.08781917380810147
Synthetic JS Distance	0.16450586525420618	0.16928093074548106
% Genome Enriched	39.16064230568816	39.57311388088635
Diff. Enrichment	10.44093839234832	10.958012116329318
CHANCE Divergence	0.08891050892690862	0.0932611193955839

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	0.5111859709254128	0.49101830140167185	0.3774548609582149	0.4809072162426557	0.38116053733376304	0.4630631908422192	0.4911634222114865	0.5067788648925522	0.5058340406823514

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	0.34557429709545046	0.1847032320554034	0.274816690788002	0.3593897757039808

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	0.03904573146044728	0.02768506602426867	0.03267941110541106	0.041348140398805425

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