QC Report

	general
Report generated at	2021-08-30 15:24:03
Title	flt-1_RW12255_midembryonic_1_5
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	24161422	18811088	18762220
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	24112718	18758020	18746530
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	99.8	99.7	99.9
Paired Reads	24161422	18811088	18762220
Paired Reads (QC-failed)	0	0	0
Read1	12080711	9405544	9381110
Read1 (QC-failed)	0	0	0
Read2	12080711	9405544	9381110
Read2 (QC-failed)	0	0	0
Properly Paired Reads	23989754	18602146	18690016
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	99.3	98.9	99.6
With itself	24102216	18745856	18739156
With itself (QC-failed)	0	0	0
Singletons	10502	12164	7374
Singletons (QC-failed)	0	0	0
% Singleton	0.0	0.1	0.0
Diff. Chroms	6902	4446	14197
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	10834522	8364014	8591277
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	1325876	1101568	779079
Paired Optical Duplicate Reads	76722	54873	53342
% Duplicate Reads	12.237499999999999	13.1703	9.0683

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	19017292	14524892	15624396
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	19017292	14524892	15624396
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	19017292	14524892	15624396
Paired Reads (QC-failed)	0	0	0
Read1	9508646	7262446	7812198
Read1 (QC-failed)	0	0	0
Read2	9508646	7262446	7812198
Read2 (QC-failed)	0	0	0
Properly Paired Reads	19017292	14524892	15624396
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	19017292	14524892	15624396
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	10759123	8306964	8540981
Distinct Fragments	9449807	7232125	7767843
Positions with Two Read	1061411	849748	671680
NRF = Distinct/Total	0.878306	0.87061	0.909479
PBC1 = OneRead/Distinct	0.875342	0.867931	0.907283
PBC2 = OneRead/TwoRead	7.793221	7.386881	10.492543

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	74107	2633
N1	58678	1543
N2	32124	1142
Np	73634	2849
N optimal	74107	2849
N conservative	74107	2633
Optimal Set	rep1_vs_rep2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0064236629817747	1.0820357007216104
Self Consistency Ratio	1.826609388619101	1.351138353765324
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	114086	125707

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	59.0	74.0	74.0
25 percentile	284.0	236.0	296.0	296.0
50 percentile (median)	284.0	236.0	296.0	296.0
75 percentile	284.0	236.0	296.0	296.0
Max size	549.0	452.0	642.0	642.0
Mean	283.725312483565	235.8293492009196	281.0972270972271	295.41450875086025

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	10142303	7832803
Estimated Fragment Length	145	125
Cross-correlation at Estimated Fragment Length	0.762298706634872	0.711778432376071
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.7616379	0.7108411
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.7551244	0.7034683
NSC (Normalized Strand Cross-correlation coeff.)	1.009501	1.011813
RSC (Relative Strand Cross-correlation coeff.)	1.101452	1.127141

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	0.40357913118238953	0.39661794847281273
Synthetic AUC	0.49853483309674085	0.4982870656112633
X-intercept	0.019009683537985426	0.019572726365179195
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5425576519916142	0.5406409104522312
Synthetic Elbow Point	0.4987397214422876	0.5023276780729989
JS Distance	0.04394701354359219	0.050974071535363465
Synthetic JS Distance	0.14798315130963474	0.15598416166262877
% Genome Enriched	36.18089248277928	36.25177198762104
Diff. Enrichment	7.499155096939569	8.328769825207027
CHANCE Divergence	0.06404306073032301	0.07105210021784934

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	0.48244818452595667	0.4886370239448252	0.46436895431799646	0.36023441688929597	0.4878980666648017	0.35311216083396696	0.4677103911897925	0.4869155210644602	0.4860301285092229

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	0.3409585374643464	0.28403386770314093	0.16232754088636253	0.3387461591648296

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	0.029349013170996856	0.020923220824500145	0.01718498147869189	0.030895960740063913

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