QC Report

	general
Report generated at	2023-07-21 17:13:56
Title	unc-4_PHX1658_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	12830022	6563218	17190712
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	11198964	5706031	14797535
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	87.3	86.9	86.1
Paired Reads	12830022	6563218	17190712
Paired Reads (QC-failed)	0	0	0
Read1	6415011	3281609	8595356
Read1 (QC-failed)	0	0	0
Read2	6415011	3281609	8595356
Read2 (QC-failed)	0	0	0
Properly Paired Reads	11141488	5676428	14661854
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	86.8	86.5	85.3
With itself	11183742	5699468	14759416
With itself (QC-failed)	0	0	0
Singletons	15222	6563	38119
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.2
Diff. Chroms	8476	3201	44959
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	5109274	2613682	6711747
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	500507	255763	672647
Paired Optical Duplicate Reads	31671	17668	50656
% Duplicate Reads	9.796000000000001	9.785499999999999	10.0219

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	9217534	4715838	12078200
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	9217534	4715838	12078200
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	9217534	4715838	12078200
Paired Reads (QC-failed)	0	0	0
Read1	4608767	2357919	6039100
Read1 (QC-failed)	0	0	0
Read2	4608767	2357919	6039100
Read2 (QC-failed)	0	0	0
Properly Paired Reads	9217534	4715838	12078200
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	9217534	4715838	12078200
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	5065545	2577006	6652840
Distinct Fragments	4570326	2325813	5990394
Positions with Two Read	418848	213778	563411
NRF = Distinct/Total	0.902238	0.902525	0.900427
PBC1 = OneRead/Distinct	0.900417	0.900382	0.898069
PBC2 = OneRead/TwoRead	9.825037	9.795765	9.5486

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	18828	117
N1	27675	91
N2	22998	65
Np	19792	144
N optimal	19792	144
N conservative	18828	117
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.051200339919269	1.2307692307692308
Self Consistency Ratio	1.2033655100443517	1.4
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	66800	65015

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	167.0	96.0	74.0	74.0
25 percentile	284.0	300.0	296.0	296.0
50 percentile (median)	284.0	300.0	296.0	296.0
75 percentile	284.0	300.0	296.0	296.0
Max size	284.0	473.0	360.0	360.0
Mean	283.9959730538922	299.9797123740675	292.5833333333333	295.9751414713015

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	4778556	2434151
Estimated Fragment Length	195	170
Cross-correlation at Estimated Fragment Length	0.609445747466413	0.446949655403337
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.6093816	0.4480291
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.6062814	0.4426593
NSC (Normalized Strand Cross-correlation coeff.)	1.005219	1.009692
RSC (Relative Strand Cross-correlation coeff.)	1.020697	0.7989741

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	0.4151861964900545	0.394805966470649
Synthetic AUC	0.4979011163090943	0.49706890228631123
X-intercept	0.019185403685800847	0.020458709370971027
Synthetic X-intercept	0.0	0.0
Elbow Point	0.4995270008661672	0.5215404204702849
Synthetic Elbow Point	0.50366073380324	0.5041063503461229
JS Distance	0.02305371551491072	0.055429754956766907
Synthetic JS Distance	0.13049465965660728	0.15863326163302327
% Genome Enriched	45.253044557715874	43.99630382111452
Diff. Enrichment	8.49682301202137	11.45444166929877
CHANCE Divergence	0.0722916636123318	0.09751845908934108

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	0.3305308122541235	0.3706511970937085	0.4004857697328223	0.4558360758634729	0.40291414231054473	0.45385971861616903	0.16591001804875374	0.34249904689287075	0.34978247901010007

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	0.10477750827294355	0.1476921050684489	0.14820250398762638	0.11134576755720008

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	0.00500797653288809	0.003743734495582007	0.009579845618106474	0.007524739883496974

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