whole_genome_sequencing

Whole genome sequencing workflow. Based on code from Chloe Girard and Melissa Pickett

File structure in the cloud

Data folder: /labs/PI_SUNetID/your_name/your_experiment

Script folder: /labs/PI_SUNetID/your_name/scripts

Load files to the cluster

1. Load files to SCG Genomics Cluster using sftp

• Make sure your .fastq files are unzipped.
• Put the files in a directory with your name and a subdirectory with your experiment (eg your_name/your_experiment)

2. Upload reference sequence in the same directory

• This should be a .tar.gz file.

Prepare reference sequence

This can go in the folder for your experiment

1. Unzip it with the command: tar xvzf file.tar.gz
2. Convert it to a dict file

module add picard
picard CreateSequenceDictionary R=genome.fa O=genome.dict
module load samtools
samtools faidx genome.fa

Upload analysis script to your scripts folder

/labs/PI_SUNetID/your_name/scripts

Run job

Submit batch to slurm:

sbatch analysis_script_location read_1.fastq read_2.fastq output_name  reference_genome_location

For example:

sbatch ../scripts/generate_hq-vcf.sh Adam-TAAGGCGA-ATAGAGAG_S8_R1_001.fastq Adam-TAAGGCGA-ATAGAGAG_S8_R2_001.fastq CB1267  ./Caenorhabditis_elegans/Ensembl/WBcel235/Sequence/Bowtie2Index/genome

Note there is no file extension at the end of the reference genome filename

Wait for the script to run on the cluster!

If you're doing a mutagenesis screen, you'll have to add some steps here to remove mutations present in your parent strain.

Export data

Add modules to your .bashrc on SCG - enter command cat>~/.bashrc - add modules to your account by pasting these lines after starting cat: module add bowtie/2.0.5 module add samtools module add gatk/3.6 module add picard module add python module add fastqc module add bedtools module add vcftools/0.1.13 export PICARD_DIR='/srv/gsfs0/software/picard' module add java/8u40 module add snpeff/4.3i - ctrl-c to quit out of cat

Add scripts for analysis - upload bash shell script that actually runs the analysis