Difference between revisions of "ChIP-Seq Top2 peak-calling E2"
| Line 111: | Line 111: | ||
== Adaptor-cutting and filtering procedure == | == Adaptor-cutting and filtering procedure == | ||
| − | cutadapt applied first (this is advised) and then trimmomatic | + | The program '''cutadapt''' was applied first (this is advised) and then '''trimmomatic''' in the following manner |
| + | |||
| + | cutadapt -g ACACTCTTTCCCTACACGACGCTCTTCCGATCT...GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG ${SAMP} -o ${OUTDIR} | ||
| + | RL="ILLUMINACLIP:${TS3PE2}:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:49" | ||
| + | java -jar $TRIMMOJARFILE PE -threads $THRDS -phred33 $SAMP1 $SAMP2 $OUTR1 $OUTSING1 $OUTR2 $OUTSING2 $RL | ||
The integrated MultiQC report is as follows: | The integrated MultiQC report is as follows: | ||
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/multiqc_report.html MultiQC]. |
The individual FastQC reports are as follows: | The individual FastQC reports are as follows: | ||
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_INP_R1_cutad_q30l49_tro_fastqc.html ULS1_INP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_INP_R2_cutad_q30l49_tro_fastqc.html ULS1_INP_R2] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_IP_R1_cutad_q30l49_tro_fastqc.html ULS1_IP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_IP_R2_cutad_q30l49_tro_fastqc.html ULS1_IP_R2] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_T2_INP_R1_cutad_q30l49_tro_fastqc.html ULS1_T2_INP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_T2_INP_R2_cutad_q30l49_tro_fastqc.html ULS1_T2_INP_R2] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_T2_IP_R1_cutad_q30l49_tro_fastqc.html ULS1_T2_IP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/ULS1_T2_IP_R2_cutad_q30l49_tro_fastqc.html ULS1_T2_IP_R2] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/WT_T2_INP_R1_cutad_q30l49_tro_fastqc.html WT_T2_INP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/WT_T2_INP_R2_cutad_q30l49_tro_fastqc.html WT_T2_INP_R2] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/WT_T2_IP_R1_cutad_q30l49_tro_fastqc.html WT_T2_IP_R1] |
| − | * [http://stab.st-andrews.ac.uk/top2/ | + | * [http://stab.st-andrews.ac.uk/top2/ffqc2/WT_T2_IP_R2_cutad_q30l49_tro_fastqc.html WT_T2_IP_R2] |
| − | |||
| − | |||
Revision as of 06:50, 21 July 2017
Contents
Introduction
Experiment 2, or the second part of Top2 - ULS1 ChIP-Seq ipeak-calling
One of the authors also has a tutorial which is formatted into wiki format (for easy copy-pasting) here.
Raw data
- ULS1 Immunoprecipitation files:
2017_06_27_07_Uls1IP-50925901 ├── 2017-06-27-07-Uls1IP_S7_L001_R1_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L001_R2_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L002_R1_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L002_R2_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L003_R1_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L003_R2_001.fastq.gz ├── 2017-06-27-07-Uls1IP_S7_L004_R1_001.fastq.gz └── 2017-06-27-07-Uls1IP_S7_L004_R2_001.fastq.gz
- ULS1 Input files
2017_06_27_06_Uls1INP-50917952 ├── 2017-06-27-06-Uls1INP_S6_L001_R1_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L001_R2_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L002_R1_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L002_R2_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L003_R1_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L003_R2_001.fastq.gz ├── 2017-06-27-06-Uls1INP_S6_L004_R1_001.fastq.gz └── 2017-06-27-06-Uls1INP_S6_L004_R2_001.fastq.gz
- Top2 Wildtype Immunoprecipitation files
2017_06_27_09_T2WTIP-50922924 ├── 2017-06-27-09-T2WTIP_S9_L001_R1_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L001_R2_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L002_R1_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L002_R2_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L003_R1_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L003_R2_001.fastq.gz ├── 2017-06-27-09-T2WTIP_S9_L004_R1_001.fastq.gz └── 2017-06-27-09-T2WTIP_S9_L004_R2_001.fastq.gz
- Top2 Wildtype Input file
2017_06_27_10_T2Uls1INP-50926896 ├── 2017-06-27-10-T2Uls1INP_S10_L001_R1_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L001_R2_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L002_R1_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L002_R2_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L003_R1_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L003_R2_001.fastq.gz ├── 2017-06-27-10-T2Uls1INP_S10_L004_R1_001.fastq.gz └── 2017-06-27-10-T2Uls1INP_S10_L004_R2_001.fastq.gz
- Top2 ULS1 Immunoprecipitation files:
2017_06_27_11_T2Uls1IP-50919923 ├── 2017-06-27-11-T2Uls1IP_S11_L001_R1_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L001_R2_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L002_R1_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L002_R2_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L003_R1_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L003_R2_001.fastq.gz ├── 2017-06-27-11-T2Uls1IP_S11_L004_R1_001.fastq.gz └── 2017-06-27-11-T2Uls1IP_S11_L004_R2_001.fastq.g
- Top2 ULS1 Input files
2017_06_27_08_T2WTINP-50917953 ├── 2017-06-27-08-T2WTINP_S8_L001_R1_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L001_R2_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L002_R1_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L002_R2_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L003_R1_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L003_R2_001.fastq.gz ├── 2017-06-27-08-T2WTINP_S8_L004_R1_001.fastq.gz └── 2017-06-27-08-T2WTINP_S8_L004_R2_001.fastq.gz
Concatenation
Splitting the rawdata into lanes is not really necessary for these experiments, so the above were concatenated to form:
rawcatdata/ ├── ULS1_INP_R1.fastq.gz ├── ULS1_INP_R2.fastq.gz ├── ULS1_IP_R1.fastq.gz ├── ULS1_IP_R2.fastq.gz ├── ULS1_T2_INP_R1.fastq.gz ├── ULS1_T2_INP_R2.fastq.gz ├── ULS1_T2_IP_R1.fastq.gz ├── ULS1_T2_IP_R2.fastq.gz ├── WT_T2_INP_R1.fastq.gz ├── WT_T2_INP_R2.fastq.gz ├── WT_T2_IP_R1.fastq.gz └── WT_T2_IP_R2.fastq.gz
(order not respected)
Dataset quality analysis and filtering
Raw unfiltered data
Using FASTQC to view quality aspects inidvidually:
- ULS1_INP_R1
- ULS1_INP_R2
- ULS1_IP_R1
- ULS1_IP_R2
- ULS1_T2_INP_R1
- ULS1_T2_INP_R2
- ULS1_T2_IP_R1
- ULS1_T2_IP_R2
- WT_T2_INP_R1
- WT_T2_INP_R2
- WT_T2_IP_R1
- WT_T2_IP_R2
and then MultiQC to integrate the FASTQC files into one report
Adaptor-cutting and filtering procedure
The program cutadapt was applied first (this is advised) and then trimmomatic in the following manner
cutadapt -g ACACTCTTTCCCTACACGACGCTCTTCCGATCT...GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG ${SAMP} -o ${OUTDIR}
RL="ILLUMINACLIP:${TS3PE2}:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:49"
java -jar $TRIMMOJARFILE PE -threads $THRDS -phred33 $SAMP1 $SAMP2 $OUTR1 $OUTSING1 $OUTR2 $OUTSING2 $RL
The integrated MultiQC report is as follows:
The individual FastQC reports are as follows:
