alignment_tutorial.md

Alignment

Connect to the server

Please replace username with your uid provided in Slack

ssh username@comp1.genomic.abi.am

Go to the working directory

cd /mnt/user/username

Create a folder for the practice and enter the folder

mkdir alignment_task
cd alignment_task

Create project folders

mkdir ref_genome # directory to store reference genome and its index files
mkdir data # directory to store fastq files
mkdir aln_res # directory to store alignment results

Download and process required files for alignment

Download fastq files with SRA toolkit

cd data
fastq-dump --gzip --skip-technical --split-files --clip SRR11881059

Perform fastqc on downloaded files

fastqc *

Trim illumina adapters

mkdir trimmed
cd trimmed
cutadapt -a AGATCGGAAGAG -A AGATCGGAAGAG -o SRR11881059_1_trimmed.fastq.gz -p SRR11881059_2_trimmed.fastq.gz ../SRR11881059_1.fastq.gz ../SRR11881059_2.fastq.gz

Run fastqc again

fastqc *

copy reference genome

cd ../../ref_genome
cp /mnt/proj/omicss24/alignment/ref_genome/GCA_000146045.2_R64_genomic.fna .

index reference genome

cd ../ref_genome
bwa index GCA_000146045.2_R64_genomic.fna
cd ..

Perform alignment with bwa mem

cd aln_res
bwa mem ../ref_genome/GCA_000146045.2_R64_genomic.fna -t 2 \
../data/trimmed/SRR11881059_1_trimmed.fastq.gz \
../data/trimmed/SRR11881059_2_trimmed.fastq.gz > aln.sam

Sort the sam file and convert it to bam

samtools sort aln.sam -@2 -o aln_sorted.bam

Create an index of a bam file

samtools index aln_sorted.bam