Assemble transcriptome and calculate gene/transcript abundance

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

yaml 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: - _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 - mergedtranscriptome.gtf - non-redundant set of transcripts - results/{SRA}/{SRA}.gtf and results/{SRA}/geneabundance.tab - re-estimated abundances for each sample - {SRA}/geneabundance{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