After perform a DEG (differential expression analysis) with DESEQ2 (Alignement with STAR, counts reads with featureCounts), I wanted to perform this time the same analysis but at the scale of isoforms with DEXSEQ.
I have 150bp paired-ends reads from two conditions (3 controls and 3 Arabidopsis mutants for one gene) that I previously aligned with STAR.
The first python script use by DEXSEQ dexseq_prepare_annotation.py) to prepare a GFF file from my GTF run successfully
But the second script doesn't work well
When I give my bam files like this:
python2 dexseq_count.py -p yes -s yes -r pos -f bam Reference/reference.DEXSeq.gff alignement.sorted.bam > counts.tsv
I have the following error message:
"If you are using alignment files in a bam format, please update your HTSeq to 0.5.4p4 or higher"
I don't understand why I have this error message because when I import htseq in python to see the version, it's indicate version 0.11.2 ..
I have partly solved the problem by converting bam files to sam files on the fly and indicate the option -f sam, the script perform well but now I have strange results , I have many eons with 0 counts, for exemple for the first gene:
AT1G01010:001 0 AT1G01010:002 0 AT1G01010:003 0 AT1G01010:004 0 AT1G01010:005 0 AT1G01010:006 0
While for the gene level I have many count:
Did someone have an explication to my problem ?
Thank's in advance