QC Report

	general
Report generated at	2021-03-26 18:00:55
Title	myc_a549
Description	chipseq of myc_a549
Pipeline version	v1.3.6
Pipeline type	tf
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': False}, 'rep2': {'paired_end': False}, 'ctl1': {'paired_end': False}, 'ctl2': {'paired_end': False}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2	ctl1	ctl2
Total Reads	31342702	29270928	24114469	28739630
Total Reads (QC-failed)	0	0	0	0
Duplicate Reads	0	0	0	0
Duplicate Reads (QC-failed)	0	0	0	0
Mapped Reads	30422844	28720855	23866522	27471733
Mapped Reads (QC-failed)	0	0	0	0
% Mapped Reads	97.1	98.1	99.0	95.6
Paired Reads	0	0	0	0
Paired Reads (QC-failed)	0	0	0	0
Read1	0	0	0	0
Read1 (QC-failed)	0	0	0	0
Read2	0	0	0	0
Read2 (QC-failed)	0	0	0	0
Properly Paired Reads	0	0	0	0
Properly Paired Reads (QC-failed)	0	0	0	0
% Properly Paired Reads	0.0	0.0	0.0	0.0
With itself	0	0	0	0
With itself (QC-failed)	0	0	0	0
Singletons	0	0	0	0
Singletons (QC-failed)	0	0	0	0
% Singleton	0.0	0.0	0.0	0.0
Diff. Chroms	0	0	0	0
Diff. Chroms (QC-failed)	0	0	0	0

Marking duplicates (filtered BAM)

	rep1	rep2	ctl1	ctl2
Unpaired Reads	24638061	23616492	19636299	22191270
Paired Reads	0	0	0	0
Unmapped Reads	0	0	0	0
Unpaired Duplicate Reads	1255818	3523253	3255994	1007565
Paired Duplicate Reads	0	0	0	0
Paired Optical Duplicate Reads	0	0	0	0
% Duplicate Reads	5.0971	14.918600000000001	16.5815	4.5404

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	ctl2
Total Reads	24638061	23616492	19636299	22191270
Total Reads (QC-failed)	0	0	0	0
Duplicate Reads	0	0	0	0
Duplicate Reads (QC-failed)	0	0	0	0
Mapped Reads	24638061	23616492	19636299	22191270
Mapped Reads (QC-failed)	0	0	0	0
% Mapped Reads	100.0	100.0	100.0	100.0
Paired Reads	0	0	0	0
Paired Reads (QC-failed)	0	0	0	0
Read1	0	0	0	0
Read1 (QC-failed)	0	0	0	0
Read2	0	0	0	0
Read2 (QC-failed)	0	0	0	0
Properly Paired Reads	0	0	0	0
Properly Paired Reads (QC-failed)	0	0	0	0
% Properly Paired Reads	0.0	0.0	0.0	0.0
With itself	0	0	0	0
With itself (QC-failed)	0	0	0	0
Singletons	0	0	0	0
Singletons (QC-failed)	0	0	0	0
% Singleton	0.0	0.0	0.0	0.0
Diff. Chroms	0	0	0	0
Diff. Chroms (QC-failed)	0	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	ctl2
Total Fragments	24537101	23531060	19514491	22021952
Distinct Fragments	23361174	20073304	16359396	21162204
Positions with Two Read	1070428	2683405	2406024	783699
NRF = Distinct/Total	0.952076	0.853056	0.83832	0.96096
PBC1 = OneRead/Distinct	0.952183	0.848579	0.83155	0.961553
PBC2 = OneRead/TwoRead	20.780568	6.347824	5.653998	25.964791

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	20692	5065
N1	12667	2201
N2	22571	6333
Np	24371	7058
N optimal	24371	7058
N conservative	20692	5065
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.1777981828726078	1.3934846989141165
Self Consistency Ratio	1.781874161206284	2.8773284870513405
Reproducibility Test	pass	borderline

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	109355	213063

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

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	110.0	120.0	115.0	115.0
25 percentile	110.0	120.0	283.0	173.0
50 percentile (median)	117.0	120.0	406.0	244.0
75 percentile	159.0	158.0	585.0	373.0
Max size	1664.0	2106.0	2201.0	2201.0
Mean	152.00473686616982	156.9677325485889	465.34981581184473	306.40068113741745

Enrichment / Signal-to-noise ratio

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

	rep1	rep2
Number of Subsampled Reads	15000000	15000000
Estimated Fragment Length	110	120
Cross-correlation at Estimated Fragment Length	0.171839714435577	0.158830985904057
Phantom Peak	35	35
Cross-correlation at Phantom Peak	0.1716357	0.1554274
Argmin of Cross-correlation	1500	1500
Minimum of Cross-correlation	0.1664778	0.1482572
NSC (Normalized Strand Cross-correlation coeff.)	1.032208	1.07132
RSC (Relative Strand Cross-correlation coeff.)	1.039557	1.474676

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	0.27258569100525243	0.2597962910710016
Synthetic AUC	0.48821504425483553	0.48797918431437626
X-intercept	0.13925176841288692	0.15705785679154521
Synthetic X-intercept	7.589384363165059e-61	1.92450703890303e-58
Elbow Point	0.5415821947113558	0.5618834546930419
Synthetic Elbow Point	0.5087683603293838	0.49450338477712696
JS Distance	0.06018551472507332	0.07119279369748464
Synthetic JS Distance	0.2551629423511463	0.269050840830157
% Genome Enriched	34.968990295221985	33.37332005774083
Diff. Enrichment	16.44341332952272	20.160254217487584
CHANCE Divergence	0.14207384018656238	0.1735019513837609

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.04770886799898742	0.09337496017613454	0.045780061759727694	0.08143046816605955	0.045680950529384215	0.08156401890678767	0.043800426459240024	0.056132069938932605	0.0560801807879182

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	0.027149935468265555	0.016475850108496767	0.03347906200463642	0.029282480349574473

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	0.014195095745680206	0.0060593648177102895	0.019715036424546032	0.017008757702097043

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