RNA-seq FAQs

Published

June 26, 2025

Modified

June 26, 2025

nfcore/rnaseq vs nfcore/differentialabundance

nfcore/rnaseq is used for pre-processing, specifically generating a count table from the FASTQ files. nfcore/differentialabundance is then used for the differential expression/abundance analysis, that is comparing expression of genes across different conditions.

Count file contains floating point numbers

The count file is generated by nfcore/rnaseq using the default path (aligner: STAR, quantification by: Salmon). Salmon “infers” (aggregates) gene counts (actually count estiamtes) from transcript-level count (estimates) and takes gene lengths into account (the gene lengths might differ across samples since not all isoforms could be expressed in a given samples). All in all the resulting count matrix contains floating point numbers as these are just estimates. One can simply round() if feeding the count table to DESeq2, for example nfcore/differentialabundance rounds the count matrix.

DESeq2 normalization

DESeq2 uses median of ratios for normalization.