QC Report

	general
Report generated at	2021-08-31 00:54:50
Title	madf-5_RW12308_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	17167086	21573336	15379328
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	17131895	21527814	15353378
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	99.8	99.8	99.8
Paired Reads	17167086	21573336	15379328
Paired Reads (QC-failed)	0	0	0
Read1	8583543	10786668	7689664
Read1 (QC-failed)	0	0	0
Read2	8583543	10786668	7689664
Read2 (QC-failed)	0	0	0
Properly Paired Reads	17080426	21461952	15301910
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	99.5	99.5	99.5
With itself	17123170	21515394	15344900
With itself (QC-failed)	0	0	0
Singletons	8725	12420	8478
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.1
Diff. Chroms	4271	7005	9850
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	7825983	9840737	7031861
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	1015074	1508870	818140
Paired Optical Duplicate Reads	94743	146455	85817
% Duplicate Reads	12.9706	15.332899999999999	11.634799999999998

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	13621818	16663734	12427442
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	13621818	16663734	12427442
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	13621818	16663734	12427442
Paired Reads (QC-failed)	0	0	0
Read1	6810909	8331867	6213721
Read1 (QC-failed)	0	0	0
Read2	6810909	8331867	6213721
Read2 (QC-failed)	0	0	0
Properly Paired Reads	13621818	16663734	12427442
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	13621818	16663734	12427442
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	7779141	9787038	6957207
Distinct Fragments	6774002	8291346	6150271
Positions with Two Read	806239	1151121	668789
NRF = Distinct/Total	0.87079	0.847176	0.884014
PBC1 = OneRead/Distinct	0.867102	0.841682	0.880556
PBC2 = OneRead/TwoRead	7.285369	6.062508	8.097712

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	92230	1124
N1	40447	337
N2	46593	325
Np	93510	1145
N optimal	93510	1145
N conservative	92230	1124
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0138783476092377	1.0186832740213523
Self Consistency Ratio	1.1519519371028755	1.0369230769230768
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	141545	142785

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	81.0	70.0	69.0	69.0
25 percentile	250.0	264.0	276.0	276.0
50 percentile (median)	250.0	264.0	276.0	276.0
75 percentile	250.0	264.0	276.0	276.0
Max size	250.0	264.0	276.0	276.0
Mean	249.99196015401463	263.9915817487831	273.64279475982534	275.9709015078601

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	7328556	9210156
Estimated Fragment Length	140	180
Cross-correlation at Estimated Fragment Length	0.694710626769698	0.732419361742284
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.6950343	0.7328616
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.6907358	0.728564
NSC (Normalized Strand Cross-correlation coeff.)	1.005754	1.005292
RSC (Relative Strand Cross-correlation coeff.)	0.9247037	0.897091

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	0.4152062174013209	0.42213617469858844
Synthetic AUC	0.49827066465499026	0.4984395815629326
X-intercept	0.019155443579782074	0.01888792618116917
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5074006492287912	0.49854662189410304
Synthetic Elbow Point	0.5004306715600064	0.5011813319535079
JS Distance	0.03274610152460587	0.029256922650352093
Synthetic JS Distance	0.12934426704660454	0.1208582357160135
% Genome Enriched	43.007414624018274	44.450611097579966
Diff. Enrichment	7.923655019290848	6.960629136547459
CHANCE Divergence	0.06733395120095456	0.059161610101854833

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	0.5550387620800689	0.560552934894424	0.4135168428301064	0.4275964285559973	0.41353384893761597	0.4305567324294462	0.5542232811209781	0.5597968879950561	0.5613371103603606

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	0.3859565115405524	0.18842580336927126	0.21276089740750784	0.39068982463981505

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	0.009136666883271602	0.0036579552009871224	0.003300460749073407	0.009212775781666453

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