QC Report

	general
Report generated at	2021-08-31 21:25:26
Title	pha-4_RW12220_L4larva_1_7
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	41848276	35094968	32449076
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	35266777	22837724	28466538
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	84.3	65.10000000000001	87.7
Paired Reads	41848276	35094968	32449076
Paired Reads (QC-failed)	0	0	0
Read1	20924138	17547484	16224538
Read1 (QC-failed)	0	0	0
Read2	20924138	17547484	16224538
Read2 (QC-failed)	0	0	0
Properly Paired Reads	35166646	22364508	28222078
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	84.0	63.7	87.0
With itself	35240234	22799930	28432894
With itself (QC-failed)	0	0	0
Singletons	26543	37794	33644
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.1
Diff. Chroms	5473	108231	92221
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	16077131	9794259	12760165
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	2689102	5361859	1254384
Paired Optical Duplicate Reads	142214	82701	102977
% Duplicate Reads	16.7263	54.744899999999994	9.8305

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	26776058	8864800	23011562
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	26776058	8864800	23011562
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	26776058	8864800	23011562
Paired Reads (QC-failed)	0	0	0
Read1	13388029	4432400	11505781
Read1 (QC-failed)	0	0	0
Read2	13388029	4432400	11505781
Read2 (QC-failed)	0	0	0
Properly Paired Reads	26776058	8864800	23011562
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	26776058	8864800	23011562
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	15790731	9664819	12663853
Distinct Fragments	13166322	4409647	11422883
Positions with Two Read	1887629	1194778	1064490
NRF = Distinct/Total	0.833801	0.456258	0.902007
PBC1 = OneRead/Distinct	0.832137	0.411051	0.899547
PBC2 = OneRead/TwoRead	5.804204	1.517094	9.652909

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	48134	1582
N1	61918	2142
N2	27350	1669
Np	59742	2947
N optimal	59742	2947
N conservative	48134	1582
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.24116009473553	1.8628318584070795
Self Consistency Ratio	2.263912248628885	1.2834032354703415
Reproducibility Test	borderline	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	100386	85172

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	74.0	86.0	85.0	85.0
25 percentile	296.0	344.0	340.0	340.0
50 percentile (median)	296.0	344.0	340.0	340.0
75 percentile	296.0	344.0	340.0	340.0
Max size	6026.0	983.0	8568.0	8568.0
Mean	296.83532564301794	343.60793453247544	377.3722429589413	341.8126276321516

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	14786068	8972366
Estimated Fragment Length	135	185
Cross-correlation at Estimated Fragment Length	0.812480208443373	0.607595811506216
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.8117316	0.5983058
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.8049928	0.5910342
NSC (Normalized Strand Cross-correlation coeff.)	1.009301	1.028021
RSC (Relative Strand Cross-correlation coeff.)	1.111085	2.27757

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	0.4013781000430325	0.371873296991188
Synthetic AUC	0.49875253744403253	0.4978151613952996
X-intercept	0.018851084221876122	0.019414160776326825
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5688411005950241	0.5917115929875005
Synthetic Elbow Point	0.5013005390095754	0.5027830586444184
JS Distance	0.05371147974898388	0.06810020733940778
Synthetic JS Distance	0.16309644085158823	0.19400397487724552
% Genome Enriched	15.937662233936344	31.530090651331815
Diff. Enrichment	5.216708635272516	9.53948010868838
CHANCE Divergence	0.05223448858820049	0.08173704089421688

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	0.4113262676679293	0.44568755076256655	0.4433996637294658	0.40266762927533617	0.44334199181537415	0.4090517552567458	0.3948463025216733	0.42776571609102587	0.42677257807724933

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	0.24824862521547603	0.28540216786205047	0.20251748488403573	0.29502022650520926

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	0.034432027422011	0.038411479389535236	0.03772200162440213	0.04627214642251317

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