Featurecounts is the fastest read summarization tool currently out there and has some great features which make it superior to HTSeq or Bedtools multicov. FeatureCounts takes GTF files as an annotation. This can be downloaded from the Ensembl FTP site . Make sure that the GTF version matches the genome that you aligned to. FeatureCounts it also smart enough to recognise and correctly process SAM and BAM alignment files. Here is a script to generate a gene-wise matrix from all BAM files in a directory. #!/bin/bash #Generate RNA-seq matrix #Set parameters GTF=/path/to/Mus_musculus.GRCm38.78.gtf EXPTNAME=mouse_rna CPUS=8 MAPQ=10 GENEMX=${EXPTNAME }_genes.mx #Make the gene-wise matrix featureCounts -Q $MAPQ -T $CPUS -a $GTF -o /dev/stdout *bam \ | cut -f1,7- | sed 1d > $GENEMX The data are now ready to analyse with your favourite statistical package (DESeq, EdgeR, Voom/Limma, etc). Consider attaching the gene name to give the data more relevance. To do that, first ...