RNA-Seq expression analysis

Question

[Ric]

Hi,

I ran successfully nf-core/rnaseq pipeline in the following way:

nextflow run nf-core/rnaseq
         -profile singularity
         -r 3.14.0
         --input samplesheet.csv
         --fasta /work/waterhouse_team/NB/LAB360/NbLab360.genome.fasta
         --save_reference
         --gff /work/waterhouse_team/NB/LAB360/NbLab360.v103.gff.gz
         --outdir results
         --aligner star_salmon
         --trimmer fastp
         --extra_fastp_args detect_adapter_for_pe,correction
         -resume

How to use the output from the above pipeline to generate:

1. A list of genes in our plant genome highly expressed in one tissue (four tissues in total), categorised by the tissue with the highest expression.
2. For each gene above, the range of expression in TPM and the ratio between the highest and average expression in other tissues in a heat map.

Thank you in advance,

Answer 1

[dthorbur]

The documentation for the pipeline shows which output directory your quantification will be in. Read these files into a tool like DESeq2 to estimate TPM.

You've given no details about how experimental design, so we cannot comment on how to organise by tissue. That said, it should be as simple as dataframe manipulation and following best practices of the tool you choose. See tutorial for DESeq2.

Both operations you've asked for help are pretty standard in rnaseq analyses. Please consult appropriate documentation, and provide a list of what you've tried to remedy the problem and where you're getting stuck when making a post.