WGBS_Methyl_seq_pipeline

Prerequisites

• BISMARK
• BOWTIE2
• SAMTOOLS

1. Preprocessing

a) using Trimmomatic

Adaptor trimming and quality filtering.

# list inputs into a file
$ cat  list_raw_name
SRR1020524
SRR1020525
SRR1020526
SRR1020527
SRR1020528
# run run_trimmomatics.sh
$ cat run_trimmomatics.sh
$SOFTPATH=<custompath>
for file in `cat list_raw_name`
do
	rm -f input_forward.fq.bz2  input_reverse.fq.bz2
	ln -s ${file}_1.fastq.bz2 input_forward.fq.bz2 
	ln -s ${file}_2.fastq.bz2 input_reverse.fq.bz2 
	#${file}
	java -jar $SOFTPATH/trimmomatic-0.36.jar PE -phred33  input_forward.fq.bz2 input_reverse.fq.bz2 output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz  ILLUMINACLIP:$SOFTPATH/adapters/TruSeq2-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
	mv output_forward_paired.fq.gz   ${file}_1_output_forward_paired.fq.gz 
	mv output_forward_unpaired.fq.gz ${file}_1_output_forward_unpaired.fq.gz
	mv output_reverse_paired.fq.gz   ${file}_2_output_reverse_paired.fq.gz
	mv output_reverse_unpaired.fq.gz ${file}_2_output_reverse_unpaired.fq.gz
done

(b) If using Trimgalore)

$ cat run_wgbs.sh 
for file in `cat list_raw_name`
do
	#trim edges using trim galore
	echo $$file
	/project/utilities/TrimGalore-0.5.0/trim_galore --paired --fastqc --trim1 ${file}_1.fastq.bz2 ${file}_2.fastq.bz2
done

2. Reference genome preparation

 FILEPATH=`pwd`
bismark_genome_preparation --bowtie2 $FILEPATH/Bisulphite-seq_HEK293/Homo_sapiens.GRCh37.75

3. Align using Bowtie2

 FILEPATH=`pwd`
/project/utilities/Bismark_v0.19.1/bismark --bowtie2 -N 1 -p 2 --parallel 2   --nucleotide_coverage  $FILEPATH/Bisulphite-seq_HEK293/Homo_sapiens.GRCh37.75 -1 SRR1020524_1_output_forward_paired.fq.gz  -2 SRR1020524_2_output_reverse_paired.fq.gz

Note: Do the same for all the replicates.

**4. Merge the alignment BAM file using SAMTOOLS **

samtools cat  -o combined_output_forward_paired_bismark_bt2_pe.bam SRR1020524_1_output_forward_paired_bismark_bt2_pe.bam SRR1020525_1_output_forward_paired_bismark_bt2_pe.bam SRR1020526_1_output_forward_paired_bismark_bt2_pe.bam SRR1020527_1_output_forward_paired_bismark_bt2_pe.bam SRR1020528_1_output_forward_paired_bismark_bt2_pe.bam 

5. Deduplication using BISMARK

/project/utilities/Bismark_v0.19.1/deduplicate_bismark -p --bam combined_output_forward_paired_bismark_bt2_pe.bam 

6.Methylation Extraction using BISMARK

/project/utilities/Bismark_v0.19.1/bismark_methylation_extractor --ignore_r2 3 --cytosine_report --CX --split_by_chromosome --gzip -p --no_overlap --ample_memory   --bedGraph --parallel 8 --genome_folder /scratch/senthil/Bisulphite-seq_HEK293/Homo_sapiens.GRCh37.75 combined_output_forward_paired_bismark_bt2_pe.deduplicated.bam