QC Report

	general
Report generated at	2021-03-26 15:46:52
Title	runx2_msc_diff
Description	chipseq of runx2_msc_diff
Pipeline version	v1.3.6
Pipeline type	tf
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': False}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1
Total Reads	40375582
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	40018846
Mapped Reads (QC-failed)	0
% Mapped Reads	99.1
Paired Reads	0
Paired Reads (QC-failed)	0
Read1	0
Read1 (QC-failed)	0
Read2	0
Read2 (QC-failed)	0
Properly Paired Reads	0
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	0.0
With itself	0
With itself (QC-failed)	0
Singletons	0
Singletons (QC-failed)	0
% Singleton	0.0
Diff. Chroms	0
Diff. Chroms (QC-failed)	0

Marking duplicates (filtered BAM)

	rep1
Unpaired Reads	34575858
Paired Reads	0
Unmapped Reads	0
Unpaired Duplicate Reads	1861465
Paired Duplicate Reads	0
Paired Optical Duplicate Reads	0
% Duplicate Reads	5.3837

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
Total Reads	34575858
Total Reads (QC-failed)	0
Duplicate Reads	0
Duplicate Reads (QC-failed)	0
Mapped Reads	34575858
Mapped Reads (QC-failed)	0
% Mapped Reads	100.0
Paired Reads	0
Paired Reads (QC-failed)	0
Read1	0
Read1 (QC-failed)	0
Read2	0
Read2 (QC-failed)	0
Properly Paired Reads	0
Properly Paired Reads (QC-failed)	0
% Properly Paired Reads	0.0
With itself	0
With itself (QC-failed)	0
Singletons	0
Singletons (QC-failed)	0
% Singleton	0.0
Diff. Chroms	0
Diff. Chroms (QC-failed)	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
Total Fragments	34575668
Distinct Fragments	32714210
Positions with Two Read	1600587
NRF = Distinct/Total	0.946163
PBC1 = OneRead/Distinct	0.947266
PBC2 = OneRead/TwoRead	19.361066

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	0	0
N1	177961	73577
Np	0	0
N optimal	177961	73577
N conservative	177961	73577
Optimal Set	rep1-pr1_vs_rep1-pr2	rep1-pr1_vs_rep1-pr2
Conservative Set	rep1-pr1_vs_rep1-pr2	rep1-pr1_vs_rep1-pr2
Rescue Ratio	0.0	0.0
Self Consistency Ratio	1.0	1.0
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
Number of peaks	327663

Top 500000 raw peaks from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	idr_opt	overlap_opt
Min size	115.0	115.0	115.0
25 percentile	136.0	268.0	195.0
50 percentile (median)	190.0	394.0	277.0
75 percentile	303.0	588.0	418.0
Max size	2466.0	2466.0	2466.0
Mean	257.79381559712266	461.34056838414176	344.1632998241188

Enrichment / Signal-to-noise ratio

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

	rep1
Number of Subsampled Reads	15000000
Estimated Fragment Length	115
Cross-correlation at Estimated Fragment Length	0.253687152866819
Phantom Peak	40
Cross-correlation at Phantom Peak	0.2355276
Argmin of Cross-correlation	1500
Minimum of Cross-correlation	0.1936754
NSC (Normalized Strand Cross-correlation coeff.)	1.309857
RSC (Relative Strand Cross-correlation coeff.)	1.433898

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
AUC	0.09139558834377712
Synthetic AUC	0.49006424904254314
X-intercept	0.41067965435475423
Synthetic X-intercept	5.031050936157443e-86
Elbow Point	0.8039330942127214
Synthetic Elbow Point	0.498546333515867
Synthetic JS Distance	0.5226412770525526

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep1-pr1	rep1-pr2
Fraction of Reads in Peaks	0.4369982084031002	0.3948470056766198	0.3947149482161802

FRiP for overlap peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.35759283254807445

FRiP for IDR peaks

	rep1-pr1_vs_rep1-pr2
Fraction of Reads in Peaks	0.25017403761896523

For macs2 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