Title: NS3104
Project: (none)
Started on: 5/11/2023 12:47:07
Hostname: login1.ufhpc
Run directory: /blue/licht/runs/Evans-MDS/NS3104/NS3104
Configuration NS3104.conf
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Table of contents:
- Input data
- Trimming and quality control
- Mapping to genome
- Genome coverage
- Peak detection
- Fraction of Reads in Peaks
- Differential peak analysis
- MultiQC report
- UCSC hub
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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: | 32D-PAR-aF-poly, 32D-Dnmt3a-F-aF-poly, 32D-PAR-aF-mono, 32D-Dnmt3a-F-aF-mono, 32D-PAR-aD-mono, 32D-Dnmt3a-F-aD-mono |
| Contrasts: | 32D-Dnmt3a-F-aF-poly vs. 32D-PAR-aF-poly, 32D-Dnmt3a-F-aF-mono vs. 32D-PAR-aF-mono, 32D-Dnmt3a-F-aD-mono vs. 32D-PAR-aD-mono |
| Number of samples | 8 |
| Sequencing data data |
| Total number of reads: | 1,750,090,567 |
| Average reads per sample: | 218,761,320 |
Table 1. Summary of input data
| Condition | Sample | Number of reads | % Reads |
| 32D-PAR-aF-poly | sP1 | 297,179,910 | 16.98% |
| 32D-Dnmt3a-F-aF-poly | sF1 | 269,341,257 | 15.39% |
| 32D-PAR-aF-mono | sP2 | 146,262,591 | 8.36% |
| 32D-Dnmt3a-F-aF-mono | sF2 | 439,316,911 | 25.10% |
| 32D-PAR-aD-mono | sP3 | 334,920,800 | 19.14% |
| 32D-Dnmt3a-F-aD-mono | sF3 | 216,933,222 | 12.40% |
Table 2. Number of reads in each sample.
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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 |
| sP1 | sP1_r1 | 297,179,910 | P1_S1_L004_R1_001
P1_S1_L004_R2_001 | 281,949,386 | P1_S1_L004_R1_001.trim.paired
P1_S1_L004_R2_001.trim.paired | 94.87% |
| sP4 | sP4_r1 | 26,321,380 | P4_S4_L004_R1_001
P4_S4_L004_R2_001 | 25,037,344 | P4_S4_L004_R1_001.trim.paired
P4_S4_L004_R2_001.trim.paired | 95.12% |
| sF1 | sF1_r1 | 269,341,257 | F1_S6_L004_R1_001
F1_S6_L004_R2_001 | 256,874,548 | F1_S6_L004_R1_001.trim.paired
F1_S6_L004_R2_001.trim.paired | 95.37% |
| sF4 | sF4_r1 | 19,814,496 | F4_S9_L004_R1_001
F4_S9_L004_R2_001 | 18,906,926 | F4_S9_L004_R1_001.trim.paired
F4_S9_L004_R2_001.trim.paired | 95.42% |
| sP2 | sP2_r1 | 146,262,591 | P2_S2_L004_R1_001
P2_S2_L004_R2_001 | 139,329,657 | P2_S2_L004_R1_001.trim.paired
P2_S2_L004_R2_001.trim.paired | 95.26% |
| sF2 | sF2_r1 | 439,316,911 | F2_S7_L004_R1_001
F2_S7_L004_R2_001 | 416,335,122 | F2_S7_L004_R1_001.trim.paired
F2_S7_L004_R2_001.trim.paired | 94.77% |
| sP3 | sP3_r1 | 334,920,800 | P3_S3_L004_R1_001
P3_S3_L004_R2_001 | 317,190,988 | P3_S3_L004_R1_001.trim.paired
P3_S3_L004_R2_001.trim.paired | 94.71% |
| sF3 | sF3_r1 | 216,933,222 | F3_S8_L004_R1_001
F3_S8_L004_R2_001 | 207,193,927 | F3_S8_L004_R1_001.trim.paired
F3_S8_L004_R2_001.trim.paired | 95.51% |
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 |
| sP1 | 297,179,910 | 281,949,386 | 94.87% |
| sP4 | 26,321,380 | 25,037,344 | 95.12% |
| sF1 | 269,341,257 | 256,874,548 | 95.37% |
| sF4 | 19,814,496 | 18,906,926 | 95.42% |
| sP2 | 146,262,591 | 139,329,657 | 95.26% |
| sF2 | 439,316,911 | 416,335,122 | 94.77% |
| sP3 | 334,920,800 | 317,190,988 | 94.71% |
| sF3 | 216,933,222 | 207,193,927 | 95.51% |
Table 4. Number of reads in each sample before and after QC.
| Condition | Reads before QC | Reads after QC | % Retained |
| 32D-PAR-aF-poly | 297,179,910 | 281,949,386 | 94.87% |
| 32D-Dnmt3a-F-aF-poly | 269,341,257 | 256,874,548 | 95.37% |
| 32D-PAR-aF-mono | 146,262,591 | 139,329,657 | 95.26% |
| 32D-Dnmt3a-F-aF-mono | 439,316,911 | 416,335,122 | 94.77% |
| 32D-PAR-aD-mono | 334,920,800 | 317,190,988 | 94.71% |
| 32D-Dnmt3a-F-aD-mono | 216,933,222 | 207,193,927 | 95.51% |
Table 5. Number of reads in each condition before and after QC.
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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 |
| sP1 | 281,949,386 | 207,042,126 | 73.43% | bam.bowtie/sP1.bt2stats.html |
| sP4 | 25,037,344 | 18,734,766 | 74.83% | bam.bowtie/sP4.bt2stats.html |
| sF1 | 256,874,548 | 185,183,422 | 72.09% | bam.bowtie/sF1.bt2stats.html |
| sF4 | 18,906,926 | 13,797,256 | 72.97% | bam.bowtie/sF4.bt2stats.html |
| sP2 | 139,329,657 | 99,997,872 | 71.77% | bam.bowtie/sP2.bt2stats.html |
| sF2 | 416,335,122 | 321,392,356 | 77.20% | bam.bowtie/sF2.bt2stats.html |
| sP3 | 317,190,988 | 240,576,234 | 75.85% | bam.bowtie/sP3.bt2stats.html |
| sF3 | 207,193,927 | 154,088,776 | 74.37% | bam.bowtie/sF3.bt2stats.html |
Table 6. Number of alignments to genome.
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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 |
| sP1 | 12,889,018,613 | 4.73 | 1,220,810,224 | 44.80% | 10.56 |
| sP4 | 122,926,804 | 0.05 | 6,180,520 | 0.20% | 19.89 |
| sF1 | 45,014,437,944 | 16.50 | 2,342,422,581 | 85.90% | 19.22 |
| sF4 | 111,786,753 | 0.04 | 3,445,059 | 0.10% | 32.45 |
| sP2 | 2,450,053,247 | 0.90 | 249,698,319 | 9.20% | 9.81 |
| sF2 | 12,188,428,838 | 4.47 | 1,022,188,971 | 37.50% | 11.92 |
| sP3 | 8,931,874,528 | 3.27 | 842,626,292 | 30.90% | 10.60 |
| sF3 | 10,150,853,675 | 3.72 | 1,007,442,695 | 36.90% | 10.08 |
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 |
| 32D-PAR-aF-poly | 12,889,018,613 | 4.73 | 1,220,810,224 | 44.80% | 10.56 |
| 32D-Dnmt3a-F-aF-poly | 45,014,437,944 | 16.50 | 2,342,422,581 | 85.90% | 19.22 |
| 32D-PAR-aF-mono | 2,450,053,247 | 0.90 | 249,698,319 | 9.20% | 9.81 |
| 32D-Dnmt3a-F-aF-mono | 0 | 0.00 | 0 | 0.00% | 0.00 |
| 32D-PAR-aD-mono | 8,931,874,528 | 3.27 | 842,626,292 | 30.90% | 10.60 |
| 32D-Dnmt3a-F-aD-mono | 10,150,853,675 | 3.72 | 1,007,442,695 | 36.90% | 10.08 |
Table 8. Genome coverage by condition
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5. Peak detection
Peak detection was performed using MACS version 2.2.7.1 with the following options: broad=N, model=Y, paired=Y, qvalue=0.05.
The following table shows the number of peaks found for each condition, and their classification. Click on the link in the Peaks column to download the list of peaks in tab-delimited format.
Table 9. Classification of peaks in genome regions
The following table provides links to the Pileup, narrowPeaks, and Summits files for each condition. All files are in bedGraph format.
Table 10. Results of peak detection with MACS.
The following histogram shows the distribution of peak locations in the different conditions.
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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 |
| 32D-PAR-aF-poly | 552,580,538 | 51,876,486 | 9.39% |
| 32D-Dnmt3a-F-aF-poly | 505,665,182 | 60,729,499 | 12.01% |
| 32D-PAR-aF-mono | 268,046,850 | 26,768,374 | 9.99% |
| 32D-Dnmt3a-F-aF-mono | 823,336,657 | 81,772,839 | 9.93% |
| 32D-PAR-aD-mono | 625,916,800 | 59,821,948 | 9.56% |
| 32D-Dnmt3a-F-aD-mono | 408,764,898 | 42,862,535 | 10.49% |
Table 11. Fraction of Reads in Peaks
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7. Differential peak analysis
For each contrast, peaks in the two conditions were compared to identify those appearing in only the test or the control, or those significantly higher in the test than in the control (abs(log2(FC)) > 1). The following table reports the number of peaks identified in each group.
Table 12. Number of differentially expressed peaks in each contrast.
The following table reports the classification of the number of differentially expressed peaks by gene region in all contrasts.
| Test | Control | Group | Upstream | Exon | CodingExon | Intron | Downstream | Intergenic |
| 32D-Dnmt3a-F-aF-poly | 32D-PAR-aF-poly | Test up | 6.32% | 3.45% | 1.15% | 60.92% | 4.60% | 23.56% |
| 32D-Dnmt3a-F-aF-poly | 32D-PAR-aF-poly | Control up | 1.56% | 1.56% | 0.00% | 56.25% | 7.81% | 32.81% |
| 32D-Dnmt3a-F-aF-poly | 32D-PAR-aF-poly | Common | 7.22% | 2.50% | 0.97% | 40.69% | 8.47% | 40.14% |
| 32D-Dnmt3a-F-aF-mono | 32D-PAR-aF-mono | Test up | 4.46% | 3.24% | 1.60% | 62.73% | 6.72% | 21.24% |
| 32D-Dnmt3a-F-aF-mono | 32D-PAR-aF-mono | Control up | 4.97% | 1.08% | 0.43% | 32.83% | 5.18% | 55.51% |
| 32D-Dnmt3a-F-aF-mono | 32D-PAR-aF-mono | Common | 5.19% | 1.89% | 1.42% | 41.09% | 7.67% | 42.74% |
| 32D-Dnmt3a-F-aD-mono | 32D-PAR-aD-mono | Test up | 4.85% | 2.61% | 1.12% | 45.52% | 6.72% | 39.18% |
| 32D-Dnmt3a-F-aD-mono | 32D-PAR-aD-mono | Control up | 3.33% | 0.48% | 0.48% | 44.76% | 4.29% | 46.67% |
| 32D-Dnmt3a-F-aD-mono | 32D-PAR-aD-mono | Common | 5.59% | 2.87% | 1.47% | 46.16% | 6.75% | 37.16% |
Table 13. Location of differential peaks for each contrast.
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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
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9. UCSC hub
UCSC Genome Browser: use the previous link to display the data tracks automatically, or copy the the URL https://bw:bw@lichtlab.cancer.ufl.edu/reports/MDS//NS3104/NS3104/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://bw:bw@lichtlab.cancer.ufl.edu/reports/MDS//NS3104/NS3104/hub.json.
Completed: 5-11-2023@12:47 |
| © 2023, A. Riva, University of Florida. |
