First follow the instructions here:

Step by step guide on how to use my pipelines
Click here for an introduction to Snakemake

ABOUT

This is a pipeline that aligns raw RNA-seq reads (downloaded from SRA) to a genome and quantifies gene abundance. Based on: Pertea, M., Kim, D., Pertea, G. M., Leek, J. T., & Salzberg, S. L. (2016). Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nature Protocols, 11(9), 1650–1667. https://doi.org/10.1038/nprot.2016.095
The final product is a table with gene abundances for every sample.

Tools used:

• fasterq-dump - download records from SRA

• BBMAP (reformat.sh) - combine paired reads into one fastq

• Hisat2 - align RNA-seq to genome

• Samtools - sort and index mapped reads

• stringtie - assemble alignments into transcripts

| | |:--:| | Pipeline workflow |

Edit config.yaml with the paths to your files

METALIST: /path/to/metalist.txt
ASSEMBLY: /path/to/assembly.fa
ANNOTATION: /path/to/annotation.gff3
OUTDIR: /path/to/outdir

• METALIST - text file with sample details. Each row is a different sample/record. Mandatory columns:

  • Run - the most important - SRA code

  • AGE - age of individual. If not applicable, leave blank or add NA

  • tissue - tissue where the sample comes from. If not applicable, leave blank or add NA

• ASSEMBLY - decompressed fasta file

• ANNOTATION - annotation gff3 file

• OUTDIR - directory where snakemake will run and where the results will be written to
  If you want the results to be written to this directory (not to a new directory), open config.yaml and comment out OUTDIR: /path/to/outdir

RESULTS

The most important results are:

• <run_date>_files.txt dated file with an overview of the files used to run the pipeline (for documentation purposes)

• reads directory that contains alignments

• results directory with final results

  • merged_transcriptome.gtf - non-redundant set of transcripts

  • results/{SRA}/{SRA}.gtf and results/{SRA}/gene_abundance.tab - re-estimated abundances for each sample

  • {SRA}/gene_abundance_{SRA}.txt - re-estimated abundances for each sample with extra informationa added - AGE and tissue

  • results/all_abundances.txt - final file with gene abundances for all samples