Sorting BAM files from HISAT2 for DEXSeq

Question

[squando]

Hi everyone, I'm new to RNA seq and still have trouble with all the nuances of bioinformatics as I do my analysis. I used HISAT2 to map all my reads to my reference genome (mouse), and have used htseq-count to continue with my differential gene expression analysis and statistics.

However, I'm also interested in looking at my reads on the exon level, and many forum posts here have suggested DEXseq. One of the run settings asks for the "Sorting order of alignments", but I can't figure out how the BAM file alignment output from HISAT2 is sorted. Should I just sort all of my BAM files using SAMtools, or does anyone happen to know how I can find out how my alignments are sorted?

Thanks!

[h.mon]

HISAT2 output is probably unsorted, you can check with:

samtools view -h file.bam | grep 'SO:'

[Arup Ghosh]

Instead than using Htseq-count try featureCounts it's much faster.

Answer 1

[Ian]

If in any doubt just rerun 'samtools sort'. Remember if you have multiple processors then use '-@ N', where N is the number of processor cores.