QC Report

	general
Report generated at	2021-09-12 14:14:08
Title	ceh-20_RW12211_L4larva_1_1
Description	mkudron
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	12523908	9884106	13951078
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	12349978	9777979	13751403
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	98.6	98.9	98.6
Paired Reads	12523908	9884106	13951078
Paired Reads (QC-failed)	0	0	0
Read1	6261954	4942053	6975539
Read1 (QC-failed)	0	0	0
Read2	6261954	4942053	6975539
Read2 (QC-failed)	0	0	0
Properly Paired Reads	12307168	9744952	13657232
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	98.3	98.6	97.89999999999999
With itself	12339420	9768592	13738020
With itself (QC-failed)	0	0	0
Singletons	10558	9387	13383
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.1
Diff. Chroms	6469	3542	34059
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	5666315	4482897	6250980
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	753628	556824	820469
Paired Optical Duplicate Reads	58383	43712	75647
% Duplicate Reads	13.3001	12.421100000000001	13.1254

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	9825374	7852146	10861022
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	9825374	7852146	10861022
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	9825374	7852146	10861022
Paired Reads (QC-failed)	0	0	0
Read1	4912687	3926073	5430511
Read1 (QC-failed)	0	0	0
Read2	4912687	3926073	5430511
Read2 (QC-failed)	0	0	0
Properly Paired Reads	9825374	7852146	10861022
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	9825374	7852146	10861022
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	5625831	4448106	6161955
Distinct Fragments	4882192	3899299	5358913
Positions with Two Read	585923	438716	637835
NRF = Distinct/Total	0.867817	0.87662	0.869677
PBC1 = OneRead/Distinct	0.864807	0.874174	0.866444
PBC2 = OneRead/TwoRead	7.205988	7.769641	7.279619

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	37418	2522
N1	30048	1249
N2	27965	1490
Np	36791	2632
N optimal	37418	2632
N conservative	37418	2522
Optimal Set	rep1_vs_rep2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0170422114103992	1.0436161776367963
Self Consistency Ratio	1.0744859645986053	1.1929543634907926
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	74988	67249

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	71.0	70.0	79.0	79.0
25 percentile	284.0	280.0	316.0	316.0
50 percentile (median)	284.0	280.0	316.0	316.0
75 percentile	284.0	280.0	316.0	316.0
Max size	284.0	280.0	426.0	426.0
Mean	283.8082626553582	279.73461315409895	302.69870820668694	315.0750708215297

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	5315430	4201190
Estimated Fragment Length	180	155
Cross-correlation at Estimated Fragment Length	0.622502523887045	0.570916908676248
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.6216566	0.5696179
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.6162312	0.5632075
NSC (Normalized Strand Cross-correlation coeff.)	1.010177	1.013688
RSC (Relative Strand Cross-correlation coeff.)	1.155913	1.202633

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	0.39656475569550836	0.3861538644776081
Synthetic AUC	0.49796941253978527	0.4977259348883882
X-intercept	0.019778307328152254	0.02008166784418446
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5587920663241887	0.5728544228765762
Synthetic Elbow Point	0.4986570144281351	0.49915916348256845
JS Distance	0.06665296890087626	0.08026179881307834
Synthetic JS Distance	0.1560080063547651	0.17021818576024403
% Genome Enriched	40.95207303005265	40.150163455435944
Diff. Enrichment	10.692065842255127	12.196832393183993
CHANCE Divergence	0.09090389254768091	0.10373000221385215

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	0.38074153716693127	0.3631537161942735	0.3839763485896112	0.3964451510593025	0.3949495245574417	0.39491456091482785	0.5007646434567745	0.36441472233328603	0.37375579238621537

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	0.22266392570903612	0.17905893455048125	0.17936523340243546	0.2197064407224543

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	0.031045361566554585	0.01762976147269305	0.022667815906632402	0.0320709579171739

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