bulkRNAseq pipeline for MSAR core at CCF

The pipeline is inspired by bulkRNAseq pipeline from bioinformatics and biostatistics core at Van andel institute.

Overview of the workflow

1. Create a samplesheet file to execute the pipeline which should be a csv file following the format below:

sample	fq1	fq2
sampleA	sampleA_R1.fastq.gz	sampleA_R2.fastq.gz

2. Execute the pipeline. Following steps/tools will be executed.
   1. fastqc on each sample - raw fastq files
   2. fastp to trim adapter sequences and low quality reads
      1. below options used for fastp
         • --qualified_quality_phred 20
         • --adapter_fasta $adapter
   3. fastq_screen on R1 fastq files to detect possible contaminants.
   4. preseq for library complexity
   5. qualimap for gene body coverage plot
   6. sortMeRNA for rRNA detection
3. multiqc for summarizing the output files of the qc tools
4. Reads alignment using STAR with quantMode GeneCounts option to generate a gene count matrix
5. Transcription quantification using Salmon

How to execute the pipeline

Adjust the configuration files such as bulk_rnaseq_conf/run.config and cluster.config. After that,

sbatch run_bulk_rnaseq.slurm

Configuration

• clustter configuration -> cluster.config
• location of reference genome -> reference.config, STAR and salmon used.
• singularity image file path -> processes.config
• run.config for location of samplesheet and turn on/off Ribodetector for rRNA removal, salmon and tpm calculator

Miscellaneous

strand info: try https://github.com/signalbash/how_are_we_stranded_here

https://github.com/igordot/genomics/blob/master/notes/rna-seq-strand.md

reverse strand for Illumina TruSeq Stranded Total RNA

https://dbrg77.wordpress.com/2015/03/20/library-type-option-in-the-tuxedo-suite/