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Cut&Run - Alignment and peak finding

Title: PHD_CR_20230612
Project: (none)
Started on: 6/14/2023 12:48:36
Hostname: login1.ufhpc
Run directory: /blue/licht/runs/MuscoLabCollab/PHD_CR_20230612/PHD_CR_20230612
Configuration PHD_CR_20230612.conf

Table of contents:

Input data
Trimming and quality control
Mapping to genome
Genome coverage
Peak detection
Fraction of Reads in Peaks
Differential peak detection
MultiQC report
UCSC hub

1. Input data
The following table summarizes the samples, conditions, and contrasts in this analysis. A readset is either a single fastq file or a pair of fastq files (for paired-end sequencing).

Category	Data
*Summary of input data*
Experimental conditions:	WT-H3K27me3, WT-H3K4me3, D1240A-H3K27me3, D1240A-H3K4me3, F1295A-H3K27me3, F1295A-H3K4me3, TKO-H3K27me3, TKO-H3K4me3
Contrasts:	WT-H3K27me3 vs. TKO-H3K27me3, D1240A-H3K27me3 vs. TKO-H3K27me3, F1295A-H3K27me3 vs. TKO-H3K27me3, WT-H3K4me3 vs. TKO-H3K4me3, D1240A-H3K4me3 vs. TKO-H3K4me3, F1295A-H3K4me3 vs. TKO-H3K4me3
Number of samples	16
*Sequencing data data*
Total number of reads:	463,831,170
Average reads per sample:	28,989,448

Table 1. Summary of input data

Condition	Sample	Number of reads	% Reads
WT-H3K27me3	WT-H3K27me3-Rep1	28,948,570	6.24%
WT-H3K27me3	WT-H3K27me3-Rep2	28,508,992	6.15%
WT-H3K4me3	WT-H3K4me3-1	30,371,791	6.55%
D1240A-H3K27me3	D1240A-H3K27me3-Rep1	34,395,801	7.42%
D1240A-H3K27me3	D1240A-H3K27me3-Rep2	23,925,283	5.16%
D1240A-H3K4me3	D1240A-H3K4me3-1	34,366,992	7.41%
F1295A-H3K27me3	F1295A-H3K27me3-Rep1	37,263,842	8.03%
F1295A-H3K27me3	F1295A-H3K27me3-Rep2	25,093,312	5.41%
F1295A-H3K4me3	F1295A-H3K4me3-1	34,248,564	7.38%
TKO-H3K27me3	TKO-H3K27me3-Rep1	30,595,380	6.60%
TKO-H3K27me3	TKO-H3K27me3-Rep2	27,448,019	5.92%
TKO-H3K4me3	TKO-H3K4me3-1	15,375,986	3.31%

Table 2. Number of reads in each sample.

2. Trimming and quality control
The input sequences were trimmed using trimmomatic. Quality control was performed before and after trimming using FastQC. The following table provides links to the quality control reports before and after trimming, as well as the number of reads in the trimmed files.

Sample	Readset	Reads before trim	QC before trim	Reads after trim	QC after trim	% Retained
WT-H3K27me3-Rep1	WT-H3K27me3-Rep1_r1	28,948,570	WT-H3K27me3-Rep1_S7_L004_R1_001 WT-H3K27me3-Rep1_S7_L004_R2_001	27,657,729	WT-H3K27me3-Rep1_S7_L004_R1_001.trim.paired WT-H3K27me3-Rep1_S7_L004_R2_001.trim.paired	95.54%
WT-H3K27me3-Rep2	WT-H3K27me3-Rep2_r1	28,508,992	WT-H3K27me3-Rep2_S8_L004_R1_001 WT-H3K27me3-Rep2_S8_L004_R2_001	27,351,179	WT-H3K27me3-Rep2_S8_L004_R1_001.trim.paired WT-H3K27me3-Rep2_S8_L004_R2_001.trim.paired	95.94%
WT-IgG	WT-IgG_r1	22,336,225	WT-IgG_S5_L004_R1_001 WT-IgG_S5_L004_R2_001	20,663,896	WT-IgG_S5_L004_R1_001.trim.paired WT-IgG_S5_L004_R2_001.trim.paired	92.51%
WT-H3K4me3-1	WT-H3K4me3-1_r1	30,371,791	WT-H3K4me3_S6_L004_R1_001 WT-H3K4me3_S6_L004_R2_001	28,257,095	WT-H3K4me3_S6_L004_R1_001.trim.paired WT-H3K4me3_S6_L004_R2_001.trim.paired	93.04%
D1240A-H3K27me3-Rep1	D1240A-H3K27me3-Rep1_r1	34,395,801	D1240A-H3K27me3-Rep1_S11_L004_R1_001 D1240A-H3K27me3-Rep1_S11_L004_R2_001	32,979,741	D1240A-H3K27me3-Rep1_S11_L004_R1_001.trim.paired D1240A-H3K27me3-Rep1_S11_L004_R2_001.trim.paired	95.88%
D1240A-H3K27me3-Rep2	D1240A-H3K27me3-Rep2_r1	23,925,283	D1240A-H3K27me3-Rep2_S12_L004_R1_001 D1240A-H3K27me3-Rep2_S12_L004_R2_001	22,937,682	D1240A-H3K27me3-Rep2_S12_L004_R1_001.trim.paired D1240A-H3K27me3-Rep2_S12_L004_R2_001.trim.paired	95.87%
D1240A-IgG	D1240A-IgG_r1	34,113,426	D1240A-IgG_S9_L004_R1_001 D1240A-IgG_S9_L004_R2_001	32,117,807	D1240A-IgG_S9_L004_R1_001.trim.paired D1240A-IgG_S9_L004_R2_001.trim.paired	94.15%
D1240A-H3K4me3-1	D1240A-H3K4me3-1_r1	34,366,992	D1240A-H3K4me3_S10_L004_R1_001 D1240A-H3K4me3_S10_L004_R2_001	32,259,322	D1240A-H3K4me3_S10_L004_R1_001.trim.paired D1240A-H3K4me3_S10_L004_R2_001.trim.paired	93.87%
F1295A-H3K27me3-Rep1	F1295A-H3K27me3-Rep1_r1	37,263,842	F1295A-H3K27me3-Rep1_S15_L004_R1_001 F1295A-H3K27me3-Rep1_S15_L004_R2_001	35,859,404	F1295A-H3K27me3-Rep1_S15_L004_R1_001.trim.paired F1295A-H3K27me3-Rep1_S15_L004_R2_001.trim.paired	96.23%
F1295A-H3K27me3-Rep2	F1295A-H3K27me3-Rep2_r1	25,093,312	F1295A-H3K27me3-Rep2_S16_L004_R1_001 F1295A-H3K27me3-Rep2_S16_L004_R2_001	24,112,495	F1295A-H3K27me3-Rep2_S16_L004_R1_001.trim.paired F1295A-H3K27me3-Rep2_S16_L004_R2_001.trim.paired	96.09%
F1295A-IgG	F1295A-IgG_r1	33,533,122	F1295A-IgG_S13_L004_R1_001 F1295A-IgG_S13_L004_R2_001	32,048,165	F1295A-IgG_S13_L004_R1_001.trim.paired F1295A-IgG_S13_L004_R2_001.trim.paired	95.57%
F1295A-H3K4me3-1	F1295A-H3K4me3-1_r1	34,248,564	F1295A-H3K4me3_S14_L004_R1_001 F1295A-H3K4me3_S14_L004_R2_001	32,386,835	F1295A-H3K4me3_S14_L004_R1_001.trim.paired F1295A-H3K4me3_S14_L004_R2_001.trim.paired	94.56%
TKO-H3K27me3-Rep1	TKO-H3K27me3-Rep1_r1	30,595,380	TKO-H3K27me3-Rep1_S3_L004_R1_001 TKO-H3K27me3-Rep1_S3_L004_R2_001	29,187,050	TKO-H3K27me3-Rep1_S3_L004_R1_001.trim.paired TKO-H3K27me3-Rep1_S3_L004_R2_001.trim.paired	95.40%
TKO-H3K27me3-Rep2	TKO-H3K27me3-Rep2_r1	27,448,019	TKO-H3K27me3-Rep2_S4_L004_R1_001 TKO-H3K27me3-Rep2_S4_L004_R2_001	26,271,898	TKO-H3K27me3-Rep2_S4_L004_R1_001.trim.paired TKO-H3K27me3-Rep2_S4_L004_R2_001.trim.paired	95.72%
TKO-IgG	TKO-IgG_r1	23,305,865	TKO-IgG_S1_L004_R1_001 TKO-IgG_S1_L004_R2_001	21,718,413	TKO-IgG_S1_L004_R1_001.trim.paired TKO-IgG_S1_L004_R2_001.trim.paired	93.19%
TKO-H3K4me3-1	TKO-H3K4me3-1_r1	15,375,986	TKO-H3K4me3_S2_L004_R1_001 TKO-H3K4me3_S2_L004_R2_001	14,646,303	TKO-H3K4me3_S2_L004_R1_001.trim.paired TKO-H3K4me3_S2_L004_R2_001.trim.paired	95.25%

Table 3. Number of reads in input files and links to QC reports.

The following two tables report the number of reads before and after QC in each sample and in each condition.

Sample	Reads before QC	Reads after QC	% Retained
WT-H3K27me3-Rep1	28,948,570	27,657,729	95.54%
WT-H3K27me3-Rep2	28,508,992	27,351,179	95.94%
WT-IgG	22,336,225	20,663,896	92.51%
WT-H3K4me3-1	30,371,791	28,257,095	93.04%
D1240A-H3K27me3-Rep1	34,395,801	32,979,741	95.88%
D1240A-H3K27me3-Rep2	23,925,283	22,937,682	95.87%
D1240A-IgG	34,113,426	32,117,807	94.15%
D1240A-H3K4me3-1	34,366,992	32,259,322	93.87%
F1295A-H3K27me3-Rep1	37,263,842	35,859,404	96.23%
F1295A-H3K27me3-Rep2	25,093,312	24,112,495	96.09%
F1295A-IgG	33,533,122	32,048,165	95.57%
F1295A-H3K4me3-1	34,248,564	32,386,835	94.56%
TKO-H3K27me3-Rep1	30,595,380	29,187,050	95.40%
TKO-H3K27me3-Rep2	27,448,019	26,271,898	95.72%
TKO-IgG	23,305,865	21,718,413	93.19%
TKO-H3K4me3-1	15,375,986	14,646,303	95.25%

Table 4. Number of reads in each sample before and after QC.

Condition	Reads before QC	Reads after QC	% Retained
WT-H3K27me3	57,457,562	55,008,908	95.74%
WT-H3K4me3	30,371,791	28,257,095	93.04%
D1240A-H3K27me3	58,321,084	55,917,423	95.88%
D1240A-H3K4me3	34,366,992	32,259,322	93.87%
F1295A-H3K27me3	62,357,154	59,971,899	96.17%
F1295A-H3K4me3	34,248,564	32,386,835	94.56%
TKO-H3K27me3	58,043,399	55,458,948	95.55%
TKO-H3K4me3	15,375,986	14,646,303	95.25%

Table 5. Number of reads in each condition before and after QC.

3. Mapping to genome
The input sequences were aligned to the genome using Bowtie 2.4.5. The following table reports the number of aligned reads for each sample. The WIG files can be uploaded to the UCSC Genome Browser as custom tracks.

Sample	Total reads	Aligned reads	Concordant alignment rate	Bowtie2 report
WT-H3K27me3-Rep1	27,657,729	19,826,525	71.69%	bam.bowtie/WT-H3K27me3-Rep1.bt2stats.html
WT-H3K27me3-Rep2	27,351,179	19,898,837	72.75%	bam.bowtie/WT-H3K27me3-Rep2.bt2stats.html
WT-IgG	20,663,896	9,528,980	46.11%	bam.bowtie/WT-IgG.bt2stats.html
WT-H3K4me3-1	28,257,095	21,207,032	75.05%	bam.bowtie/WT-H3K4me3-1.bt2stats.html
D1240A-H3K27me3-Rep1	32,979,741	24,089,897	73.04%	bam.bowtie/D1240A-H3K27me3-Rep1.bt2stats.html
D1240A-H3K27me3-Rep2	22,937,682	16,851,350	73.47%	bam.bowtie/D1240A-H3K27me3-Rep2.bt2stats.html
D1240A-IgG	32,117,807	17,942,991	55.87%	bam.bowtie/D1240A-IgG.bt2stats.html
D1240A-H3K4me3-1	32,259,322	22,881,499	70.93%	bam.bowtie/D1240A-H3K4me3-1.bt2stats.html
F1295A-H3K27me3-Rep1	35,859,404	27,977,235	78.02%	bam.bowtie/F1295A-H3K27me3-Rep1.bt2stats.html
F1295A-H3K27me3-Rep2	24,112,495	17,727,506	73.52%	bam.bowtie/F1295A-H3K27me3-Rep2.bt2stats.html
F1295A-IgG	32,048,165	20,165,942	62.92%	bam.bowtie/F1295A-IgG.bt2stats.html
F1295A-H3K4me3-1	32,386,835	25,350,562	78.27%	bam.bowtie/F1295A-H3K4me3-1.bt2stats.html
TKO-H3K27me3-Rep1	29,187,050	21,311,437	73.02%	bam.bowtie/TKO-H3K27me3-Rep1.bt2stats.html
TKO-H3K27me3-Rep2	26,271,898	19,191,464	73.05%	bam.bowtie/TKO-H3K27me3-Rep2.bt2stats.html
TKO-IgG	21,718,413	9,788,834	45.07%	bam.bowtie/TKO-IgG.bt2stats.html
TKO-H3K4me3-1	14,646,303	9,491,694	64.81%	bam.bowtie/TKO-H3K4me3-1.bt2stats.html

Table 6. Number of alignments to genome.

4. Genome coverage
The following table reports the overall and effective genome coverage in each sample. The Total nt column reports the total number of nucleotides sequenced, i.e. the number of aligned reads times the length of each read. Coverage is this number divided by the size of the genome. Effective bp reports the number of bases in the genome having coverage greater than 5, and the Effective Perc column shows what percentage this is of the genome size. Note that, especially in the case of RNA-seq, the effective genome size may be much smaller than the full size. Eff Coverage is the average coverage over the effectively covered fraction of the genome.

Name	Total nt	Coverage	Effective bp	Effective Perc	Eff Coverage
WT-H3K27me3-Rep1	4,163,719,552	1.35	180,837,235	5.90%	23.02
WT-H3K27me3-Rep2	4,118,097,446	1.33	192,419,670	6.20%	21.40
WT-IgG	459,194,788	0.15	44,167,769	1.40%	10.40
WT-H3K4me3-1	3,056,258,731	0.99	42,228,688	1.40%	72.37
D1240A-H3K27me3-Rep1	4,955,173,927	1.61	258,672,056	8.40%	19.16
D1240A-H3K27me3-Rep2	3,305,134,621	1.07	193,159,495	6.30%	17.11
D1240A-IgG	818,929,244	0.27	89,191,993	2.90%	9.18
D1240A-H3K4me3-1	2,874,183,511	0.93	49,623,311	1.60%	57.92
F1295A-H3K27me3-Rep1	5,282,552,793	1.71	289,695,843	9.40%	18.23
F1295A-H3K27me3-Rep2	3,088,047,820	1.00	201,242,869	6.50%	15.34
F1295A-IgG	898,129,191	0.29	96,408,689	3.10%	9.32
F1295A-H3K4me3-1	3,756,192,513	1.22	51,793,863	1.70%	72.52
TKO-H3K27me3-Rep1	4,253,547,208	1.38	242,032,205	7.80%	17.57
TKO-H3K27me3-Rep2	3,661,733,903	1.19	223,610,350	7.20%	16.38
TKO-IgG	400,476,845	0.13	40,576,192	1.30%	9.87
TKO-H3K4me3-1	1,627,896,672	0.53	28,544,793	0.90%	57.03

Table 7. Genome coverage by sample.

The following table reports the overall and effective genome coverage in each condition.

Name	Total nt	Coverage	Effective bp	Effective Perc	Eff Coverage
WT-H3K27me3	9,041,109,112	2.93	305,965,930	9.90%	29.55
WT-H3K4me3	3,056,258,731	0.99	42,228,688	1.40%	72.37
D1240A-H3K27me3	9,268,999,366	3.00	387,459,458	12.60%	23.92
D1240A-H3K4me3	2,874,183,511	0.93	49,623,311	1.60%	57.92
F1295A-H3K27me3	9,563,155,396	3.10	436,935,261	14.20%	21.89
F1295A-H3K4me3	3,756,192,513	1.22	51,793,863	1.70%	72.52
TKO-H3K27me3	9,007,093,616	2.92	402,937,010	13.10%	22.35
TKO-H3K4me3	1,627,896,672	0.53	28,544,793	0.90%	57.03

Table 8. Genome coverage by condition

File: PHD_CR_20230612.sample.cov.xlsx
Size: 40.79 kB
Description: Per-chromosome coverage data, by sample.

File: PHD_CR_20230612.cond.cov.xlsx
Size: 22.89 kB
Description: Per-chromosome coverage data, by condition.

5. Peak detection
Peak detection was performed using SEACR with the following options: mode=relaxed, normalization=True. The following table shows the number of peaks found for each condition.

Condition	Total Peaks	Peaks	Actions
WT-H3K27me3	3,790	WT-H3K27me3.peaks.bed	Create RegionSet
WT-H3K4me3	8,140	WT-H3K4me3.peaks.bed	Create RegionSet
D1240A-H3K27me3	2,188	D1240A-H3K27me3.peaks.bed	Create RegionSet
D1240A-H3K4me3	7,712	D1240A-H3K4me3.peaks.bed	Create RegionSet
F1295A-H3K27me3	2,083	F1295A-H3K27me3.peaks.bed	Create RegionSet
F1295A-H3K4me3	8,239	F1295A-H3K4me3.peaks.bed	Create RegionSet
TKO-H3K27me3	5,881	TKO-H3K27me3.peaks.bed	Create RegionSet
TKO-H3K4me3	7,396	TKO-H3K4me3.peaks.bed	Create RegionSet

Table 9. Peaks detected by SEACR and their classification in genomic regions.

The following histogram shows the distribution of peak locations in the different conditions.

File: peak-classifications.xlsx
Size: 5.85 kB
Description: Table containing number of peaks in each region for each condition.

6. Fraction of Reads in Peaks
The Fraction of Reads in Peaks (FRIP) is the fraction of reads that fall in regions called as peaks, out of all aligned peaks.

Condition	Reads	Reads in peaks	FRIP
WT-H3K27me3	98,508,895	45,808,755	46.50%
WT-H3K4me3	49,504,921	33,021,110	66.70%
D1240A-H3K27me3	102,137,183	28,922,745	28.32%
D1240A-H3K4me3	54,674,630	32,223,153	58.94%
F1295A-H3K27me3	111,485,180	29,479,390	26.44%
F1295A-H3K4me3	58,245,844	37,630,536	64.61%
TKO-H3K27me3	100,081,195	38,398,511	38.37%
TKO-H3K4me3	23,100,943	15,707,557	68.00%

Table 10. Fraction of Reads in Peaks

7. Differential peak detection
The following table reports peaks that are increased, decreased, or unchanged in each contrast. Note that these results are simply based on the fold change of the peak sizes in the two conditions, with no statistical significance information, using a Log2(FC) threshold of 1.0. More accurate differential analysis can be performed with DASA.

Test	Control	Total	Increased	Decreased	Unchanged	Table
WT-H3K27me3	TKO-H3K27me3	3,801	32%	19%	47%	WT-H3K27me3.vs.TKO-H3K27me3.xlsx
D1240A-H3K27me3	TKO-H3K27me3	2,264	13%	31%	55%	D1240A-H3K27me3.vs.TKO-H3K27me3.xlsx
F1295A-H3K27me3	TKO-H3K27me3	2,290	31%	12%	56%	F1295A-H3K27me3.vs.TKO-H3K27me3.xlsx
WT-H3K4me3	TKO-H3K4me3	7,329	91%	0%	8%	WT-H3K4me3.vs.TKO-H3K4me3.xlsx
D1240A-H3K4me3	TKO-H3K4me3	7,249	91%	0%	8%	D1240A-H3K4me3.vs.TKO-H3K4me3.xlsx
F1295A-H3K4me3	TKO-H3K4me3	7,346	99%	0%	0%	F1295A-H3K4me3.vs.TKO-H3K4me3.xlsx

Show values | percentages
Table 11. Differential peak analysis results.

8. MultiQC report
MultiQC is a general Quality Control tool for a large number of bioinformatics pipelines. The report on this analysis (generated using MultiQC version 1.12) is available here:

MultiQC report

9. UCSC hub

UCSC Genome Browser: use the previous link to display the data tracks automatically, or copy the the URL https://lichtlab.cancer.ufl.edu/reports/MLC//PHD_CR_20230612/PHD_CR_20230612/hub.txt and paste it into the "My Hubs" form in this page.

WashU EpiGenome Browser: use the previous link to display the data tracks automatically, or copy the following URL into the "Datahub by URL Link" field: https://lichtlab.cancer.ufl.edu/reports/MLC//PHD_CR_20230612/PHD_CR_20230612/hub.json.

Completed: 6-14-2023@12:48