QC Report

	general
Report generated at	2021-08-31 12:03:04
Title	nhr-41_RW12221_L4larva_1_2
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	12337632	14072592	17097476
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	12090867	13956872	16769725
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	98.0	99.2	98.1
Paired Reads	12337632	14072592	17097476
Paired Reads (QC-failed)	0	0	0
Read1	6168816	7036296	8548738
Read1 (QC-failed)	0	0	0
Read2	6168816	7036296	8548738
Read2 (QC-failed)	0	0	0
Properly Paired Reads	12049170	13908684	16667146
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	97.7	98.8	97.5
With itself	12084566	13949032	16761136
With itself (QC-failed)	0	0	0
Singletons	6301	7840	8589
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.1
Diff. Chroms	4602	5059	38050
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	5520492	6380285	7597600
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	969287	1191808	1474071
Paired Optical Duplicate Reads	47211	58581	78666
% Duplicate Reads	17.558	18.679499999999997	19.401799999999998

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	9102410	10376954	12247058
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	9102410	10376954	12247058
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	9102410	10376954	12247058
Paired Reads (QC-failed)	0	0	0
Read1	4551205	5188477	6123529
Read1 (QC-failed)	0	0	0
Read2	4551205	5188477	6123529
Read2 (QC-failed)	0	0	0
Properly Paired Reads	9102410	10376954	12247058
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	9102410	10376954	12247058
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	5467723	6315973	7506576
Distinct Fragments	4509412	5139353	6053841
Positions with Two Read	701484	845517	1026961
NRF = Distinct/Total	0.824733	0.813707	0.806472
PBC1 = OneRead/Distinct	0.818062	0.805764	0.79798
PBC2 = OneRead/TwoRead	5.258818	4.897718	4.704021

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	31885	1069
N1	28634	347
N2	27684	676
Np	32947	1136
N optimal	32947	1136
N conservative	31885	1069
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0333071977418848	1.0626753975678205
Self Consistency Ratio	1.0343158503106487	1.9481268011527377
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	73868	65270

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	75.0	72.0	72.0
25 percentile	260.0	264.0	284.0	284.0
50 percentile (median)	260.0	264.0	284.0	284.0
75 percentile	260.0	264.0	284.0	284.0
Max size	260.0	264.0	284.0	284.0
Mean	259.9935425353333	263.98008273326184	279.93221830985914	283.85974443803684

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	5159065	5967825
Estimated Fragment Length	165	165
Cross-correlation at Estimated Fragment Length	0.592876464237337	0.623287490204593
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.593231	0.6232462
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.5894372	0.6190163
NSC (Normalized Strand Cross-correlation coeff.)	1.005835	1.0069
RSC (Relative Strand Cross-correlation coeff.)	0.906539	1.00977

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	0.40460357516307766	0.4042264976403156
Synthetic AUC	0.49788660015759606	0.49802064350176634
X-intercept	0.019686155221831016	0.01958236529264768
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5285781578085954	0.5370011097538582
Synthetic Elbow Point	0.49885390936620533	0.5019191416562515
JS Distance	0.043134133041918946	0.045348974376874636
Synthetic JS Distance	0.14520281853042388	0.1472821241902706
% Genome Enriched	43.91072469322092	41.97464332191644
Diff. Enrichment	9.615767938312326	9.51537839536975
CHANCE Divergence	0.08179019908936622	0.08087742011051276

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	0.3515651349477776	0.3180416912323212	0.3946927473728941	0.3753674969808102	0.39955361262646105	0.3761110198832952	0.5373060434621992	0.33457594722785666	0.3428937090335617

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	0.16614176931033273	0.15174080271049095	0.14979010218220107	0.17110984732355738

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	0.010991631964986126	0.004256565019593712	0.008019019839540582	0.01161542029811651

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