Dear All,

I am attempting to use DEXSeq version 1.14.2 to analyse alternative splicing in my RNA-seq data. Generally, I am pretty comfortable in R and try to keep my analyses in R, but the initial steps of the DEXSeq pipeline (as described in the 2015 vignette for version 1.14.2 by Reyes et al) involve a little bit of python and I'm hoping someone would help me with the error I am getting.

I have prepared the annotation file using the first of the python scripts supplied in the package, for my analysis this is the file Dmel.DEXSeq.chr.gff

When it comes to counting reads, I try to feed in my paired-end sorted bam file (in this example, sample180.bam) with this:

python /my/file/path/DEXSeq/python_scripts/dexseq_count.py -p yes -r name -f bam Dmel.DEXSeq.chr.gff sample180.bam sample180.txt

and I get the error:

KeyError: 'SAM optional field tag NH not found'

I understand the error message, and a quick internet search showed me that this error has come up before and can be solved by simply adding the NH tags into the bam file. However, with my limited python knowledge, I have no idea how to even start doing this, so I hoped someone might be kind enough to give a beginner's guide to solving this issue.

Many thanks in advance,

Fiona