version 2024

2024/ebaiin1/chip-seq/README.md

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+## EBAII 2024 - ChIP-seq course
+
+### Hands-on
+Go to [hands-on](hands-on/hands-on.html)
+
+### Planning
+
+#### Tuesday
+
+
+| **Start** | **End** | **Duration** | **Topics** | **Teacher** |
+| -------- | --------- | --------- | ----------- | ----------- |
+| 10:45 | 12:45 | 02:00 | Experimental design | Stéphanie Le Gras |
+| 12:45 | 14:30 | 01:45 | **Lunch Break** |  |
+| 14:30 | 16:00 | 01:30 | Mapping | Stéphanie Le Gras |
+| 16:00 | 16:30 | 00:30 | **Break** |  |
+| 16:30 | 17:45 | 01:15 | Mapping QC, Visualization | Tao Ye |
+| 17:45 | 19:00 | 01:15 | Mapping QC, Visualization, Peak calling | Tao Ye |
+
+
+#### Wednesday
+
+
+| **Start** | **End** | **Duration** | **Topics** | **Teacher** |
+| -------- | --------- | --------- | ----------- | ----------- |
+| 10:45 | 12:25 | 02:00 | Motif analysis | Morgane THOMAS-CHOLLIER |
+| 12:45 | 14:30 | 01:45 | **Break** |  |
+| 14:30 | 16:00 | 01:30 | Free time |
+| 16:00 | 16:30 | 00:30 | **Break** |  |
+| 16:30 | 17:45 | 01:15 | Peak annotation | Elodie Darbo |
+
+
+#### Thursday
+
+
+| **Start** | **End** | **Duration** | **Topics** | **Teacher** |
+| -------- | --------- | --------- | ----------- | ----------- |
+| 8:30 | 10:15 | 01:45 | Scripting / Workflow | Elodie Darbo |

2024/ebaiin1/chip-seq/_config.yml

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+theme: jekyll-theme-cayman

2024/ebaiin1/chip-seq/hands-on/cmd.sh

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+## Création de l'environnement
+mkdir M2.2-BIMS-epigenomique
+cd M2.2-BIMS-epigenomique
+cp -r ../EBAII2021_chipseq/data .
+
+## Controle qualité
+module add fastqc/0.11.9
+
+mkdir 01-QualityControl
+cd 01-QualityControl
+fastqc ../data/SRR576933.fastq.gz -o .
+fastqc ../data/SRR576934.fastq.gz -o .
+fastqc ../data/SRR576938.fastq.gz -o .
+cd ..
+
+## Mapping
+module add bowtie/1.2.3
+
+mkdir 02-Mapping
+cd 02-Mapping
+mkdir index
+cd index
+bowtie-build ../../data/Escherichia_coli_K12.fasta Escherichia_coli_K12
+cd ..
+mkdir bam
+cd bam
+sbatch --cpus-per-task 10 --wrap="bowtie -p 10 ../index/Escherichia_coli_K12 ../../data/SRR576938.fastq.gz -v 2 -m 1 -3 1 -S 2> SRR576938.out > SRR576938.sam"
+sbatch --cpus-per-task 10 --wrap="bowtie -p 10 ../index/Escherichia_coli_K12 ../../data/SRR576934.fastq.gz -v 2 -m 1 -3 1 -S 2> SRR576934.out > SRR576934.sam"
+sbatch --cpus-per-task 10 --wrap="bowtie -p 10 ../index/Escherichia_coli_K12 ../../data/SRR576933.fastq.gz -v 2 -m 1 -3 1 -S 2> SRR576933.out > SRR576933.sam"
+
+# Création de fichiers sam ordonnés
+module add samtools/1.10
+samtools sort SRR576933.sam | samtools view -b > SRR576933.bam
+samtools sort SRR576934.sam | samtools view -b > SRR576934.bam
+samtools sort SRR576938.sam | samtools view -b > SRR576938.bam
+
+# On index les fichiers bam
+samtools index SRR576934.bam
+samtools index SRR576933.bam
+samtools index SRR576938.bam
+
+# On compresse les fichiers sam
+gzip SRR576933.sam &
+gzip SRR576934.sam &
+gzip SRR576938.sam &
+
+# marquage des duplicats
+module add picard/2.22.0
+
+picard MarkDuplicates CREATE_INDEX=true INPUT=SRR576933.bam OUTPUT=Marked_SRR576933.bam METRICS_FILE=metrics VALIDATION_STRINGENCY=STRICT
+picard MarkDuplicates CREATE_INDEX=true INPUT=SRR576934.bam OUTPUT=Marked_SRR576934.bam METRICS_FILE=metrics VALIDATION_STRINGENCY=STRICT
+picard MarkDuplicates CREATE_INDEX=true INPUT=SRR576938.bam OUTPUT=Marked_SRR576938.bam METRICS_FILE=metrics VALIDATION_STRINGENCY=STRICT
+
+cd ../..
+
+## Control qualité
+module add deeptools/3.2.0
+
+mkdir 03-ChIPQualityControls
+cd 03-ChIPQualityControls
+plotFingerprint --numberOfSamples 10000 -b ../02-Mapping/bam/SRR576933.bam ../02-Mapping/bam/SRR576934.bam ../02-Mapping/bam/SRR576938.bam -plot fingerprint_10000.png &
+plotFingerprint -b ../02-Mapping/bam/SRR576933.bam ../02-Mapping/bam/SRR576934.bam ../02-Mapping/bam/SRR576938.bam -plot fingerprint.png &
+
+cd ..
+
+## Génération de fichiers bigwig
+module add deeptools/3.2.0
+
+mkdir 04-Visualization
+cd 04-Visualization/
+bamCoverage --bam ../02-Mapping/bam/Marked_SRR576933.bam --outFileName SRR576933_nodup.bw --outFileFormat bigwig --effectiveGenomeSize 4639675 --normalizeUsing RPGC --skipNonCoveredRegions --extendReads 200 --ignoreDuplicates
+bamCoverage --bam ../02-Mapping/bam/Marked_SRR576934.bam --outFileName SRR576934_nodup.bw --outFileFormat bigwig --effectiveGenomeSize 4639675 --normalizeUsing RPGC --skipNonCoveredRegions --extendReads 200 --ignoreDuplicates
+bamCoverage --bam ../02-Mapping/bam/Marked_SRR576938.bam --outFileName SRR576938_nodup.bw --outFileFormat bigwig --effectiveGenomeSize 4639675 --normalizeUsing RPGC --skipNonCoveredRegions --extendReads 200 --ignoreDuplicates
+
+cd ..
+
+## Peak calling
+module add macs2/2.2.7.1
+
+mkdir 05-PeakCalling
+# Peak calling sur les réplicats
+mkdir 05-PeakCalling/replicates
+cd 05-PeakCalling/replicates
+macs2 callpeak -t ../../02-Mapping/bam/SRR576933.bam -c ../../02-Mapping/bam/SRR576938.bam --format BAM --gsize 4639675 --name 'FNR_Anaerobic_A' --bw 400 --fix-bimodal -p 1e-2 &> repA_MACS.out
+macs2 callpeak -t ../../02-Mapping/bam/SRR576934.bam -c ../../02-Mapping/bam/SRR576938.bam --format BAM --gsize 4639675 --name 'FNR_Anaerobic_B' --bw 400 --fix-bimodal -p 1e-2 &> repB_MACS.out
+cd ..
+
+# Peak calling sur le pool de réplicat
+mkdir pool
+cd pool
+macs2 callpeak -t ../../02-Mapping/bam/SRR576933.bam ../../02-Mapping/bam/SRR576934.bam -c ../../02-Mapping/bam/SRR576938.bam --format BAM --gsize 4639675 --name 'FNR_Anaerobic_pool' --bw 400 --fix-bimodal -p 1e-2 &> pool_MACS.out
+cd ..
+
+# Analyse IDR
+mkdir idr
+cd idr
+idr --samples ../replicates/FNR_Anaerobic_A_peaks.narrowPeak ../replicates/FNR_Anaerobic_B_peaks.narrowPeak --peak-list ../pool/FNR_Anaerobic_pool_peaks.narrowPeak \
+--input-file-type narrowPeak --output-file FNR_anaerobic_idr_peaks.bed --plot
+cd ../..
+
+## Préparation des fichiers pour l'analyse de motif
+module add bedtools/2.29.2
+
+mkdir 06-MotifAnalysis
+cd 06-MotifAnalysis
+samtools faidx ../data/Escherichia_coli_K12.fasta
+bedtools getfasta -fi ../data/Escherichia_coli_K12.fasta \
+-bed ../05-PeakCalling/idr/FNR_anaerobic_idr_peaks.bed -fo FNR_anaerobic_idr_peaks.fa
+
+cd ..
+
+## Annotation des pics
+module add homer/4.10
+
+mkdir 07-PeakAnnotation
+cd 07-PeakAnnotation
+gunzip ../data/Escherichia_coli_K_12_MG1655.annotation.fixed.gtf.gz
+gunzip ../data/Escherichia_coli_K12.fasta.gz
+
+# On met le fichier de pics dans le bon format
+cut -f1-5 ../05-PeakCalling/idr/FNR_anaerobic_idr_peaks.bed |   awk -F "\t" '{print $0"\t+"}'  > FNR_anaerobic_idr_peaks.bed
+
+# lancement de l'annotation
+annotatePeaks.pl   FNR_anaerobic_idr_peaks.bed   ../data/Escherichia_coli_K12.fasta   -gtf ../data/Escherichia_coli_K_12_MG1655.annotation.fixed.gtf   > FNR_anaerobic_idr_annotated_peaks.tsv
+
+# calcul de statistiques sur les résultats
+tail -n 2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{print $11}'
+vi FNR_anaerobic_idr_final_peaks_annotation.tsv
+tail -n +2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{print $11}'
+tail -n +2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{print $8}' | sort | uniq -c
+tail -n +2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{if ($8=="promoter-TSS") print $11}'
+tail -n +2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{if ($8=="promoter-TSS") print $11}' | wc -l
+tail -n +2 FNR_anaerobic_idr_final_peaks_annotation.tsv | awk '{if ($8=="promoter-TSS") print $11}' > FNR_anaerobic_idr_final_peaks_annotation_officialGeneSymbols.tsv
+
+# On compresse de nouveau les fichiers
+gzip ../data/Escherichia_coli_K_12_MG1655.annotation.fixed.gtf
+gzip ../data/Escherichia_coli_K12.fasta
+
+cd ..