QC Report

	general
Report generated at	2021-08-31 00:02:30
Title	madf-5_OP436_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	14631864	16617388	17431968
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	14557341	16080149	17361717
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	99.5	96.8	99.6
Paired Reads	14631864	16617388	17431968
Paired Reads (QC-failed)	0	0	0
Read1	7315932	8308694	8715984
Read1 (QC-failed)	0	0	0
Read2	7315932	8308694	8715984
Read2 (QC-failed)	0	0	0
Properly Paired Reads	14479466	16017856	17279970
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	99.0	96.39999999999999	99.1
With itself	14538210	16066948	17347750
With itself (QC-failed)	0	0	0
Singletons	19131	13201	13967
Singletons (QC-failed)	0	0	0
% Singleton	0.1	0.1	0.1
Diff. Chroms	6426	4876	25510
Diff. Chroms (QC-failed)	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1
Unpaired Reads	0	0	0
Paired Reads	6620434	7317047	7961697
Unmapped Reads	0	0	0
Unpaired Duplicate Reads	0	0	0
Paired Duplicate Reads	773183	942716	1070828
Paired Optical Duplicate Reads	47686	56986	76273
% Duplicate Reads	11.678700000000001	12.8838	13.4497

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	11694502	12748662	13781738
Total Reads (QC-failed)	0	0	0
Duplicate Reads	0	0	0
Duplicate Reads (QC-failed)	0	0	0
Mapped Reads	11694502	12748662	13781738
Mapped Reads (QC-failed)	0	0	0
% Mapped Reads	100.0	100.0	100.0
Paired Reads	11694502	12748662	13781738
Paired Reads (QC-failed)	0	0	0
Read1	5847251	6374331	6890869
Read1 (QC-failed)	0	0	0
Read2	5847251	6374331	6890869
Read2 (QC-failed)	0	0	0
Properly Paired Reads	11694502	12748662	13781738
Properly Paired Reads (QC-failed)	0	0	0
% Properly Paired Reads	100.0	100.0	100.0
With itself	11694502	12748662	13781738
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	6574754	7275739	7843051
Distinct Fragments	5813131	6343139	6794445
Positions with Two Read	627821	746989	842453
NRF = Distinct/Total	0.884159	0.871821	0.866301
PBC1 = OneRead/Distinct	0.881037	0.868404	0.861593
PBC2 = OneRead/TwoRead	8.157712	7.374145	6.94881

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	87154	813
N1	43710	411
N2	47884	254
Np	88225	844
N optimal	88225	844
N conservative	87154	813
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.0122885926061913	1.038130381303813
Self Consistency Ratio	1.095493022191718	1.6181102362204725
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	147881	153438

Top 300000 raw peaks from spp with FDR 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	54.0	74.0	58.0	58.0
25 percentile	216.0	216.0	224.0	224.0
50 percentile (median)	216.0	216.0	224.0	224.0
75 percentile	216.0	216.0	224.0	224.0
Max size	216.0	216.0	312.0	312.0
Mean	215.99310256219528	215.9989311643791	222.3791469194313	223.9841994899405

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	6189856	6844991
Estimated Fragment Length	130	120
Cross-correlation at Estimated Fragment Length	0.662674839499644	0.679155290477171
Phantom Peak	50	50
Cross-correlation at Phantom Peak	0.6632853	0.6795972
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.6585223	0.6751187
NSC (Normalized Strand Cross-correlation coeff.)	1.006306	1.005979
RSC (Relative Strand Cross-correlation coeff.)	0.8718352	0.9013333

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	0.4092826669926271	0.41517191487590205
Synthetic AUC	0.4980985599991123	0.49818465978961574
X-intercept	0.01954791454974118	0.019412165143145754
Synthetic X-intercept	0.0	0.0
Elbow Point	0.5148755397536148	0.4979088602439896
Synthetic Elbow Point	0.5027557515438971	0.5015749085450872
JS Distance	0.04915515906001998	0.04685460980903883
Synthetic JS Distance	0.13763184560729846	0.12836438322368715
% Genome Enriched	45.328124282574365	45.76851132808835
Diff. Enrichment	8.753491253930568	8.066400151758257
CHANCE Divergence	0.07453495931556649	0.06867925095526077

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	0.5368485122325004	0.5499908931619648	0.43064776411209915	0.44892311853226347	0.43961263841977	0.4475113462905121	0.5342241699969775	0.5429409150237808	0.5452959437323162

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	0.3246422189860527	0.18695982094833966	0.19806941308821271	0.3284269172354283

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	0.006829230454780732	0.004395569815627891	0.00255140500234456	0.007076293396386818

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