Dear All,
I have some RNA paired-end files which I have used tophat to align
~/tophat-2.0.7.Linux_x86_64/./tophat -p10 --segment-length 18 \
--no-coverage-search --segment-mismatches 1 -G
then samtools
samtools sort -n /media//PRE_6_8_2/accepted_hits.bam \
/media/PRE_6_8_2/PRE_6_8_2.sort.hg19.bam
and then
samtools view /media/PRE_6_8_2/PRE_6_8_2.sort.hg19.bam.bam \
/media/PRE_6_8_2/PRE_6_8_2.sort.hg19.sam
then I am trying to use DEXSeq and using the
python2.7 /home/3.0/DEXSeq/python_scripts/dexseq_count.py -p yes -r name -s no \
/media/PRE_6_8_2/hg19/hg19_IlluminaAnnotation_genes.gtf \
/media/PRE_6_8_2/PRE_6_8_2.sort.hg19.sam \
/media/PRE_6_8_2//PRE_6_8_2/untreated1fb.txt
and gives me
_ambiguous 0
_empty 39845364
_lowaqual 0
_notaligned 0
without any reads for any exons. 0 for everything
whereas by using the htseq-count -r name -s no
everything looks fine (reads in genes)...
could you please advice me how to solve it? I really need to run the DEXseq as I want reads per exon to detect splice differences.
Thank you in advance for your help
That's the problem...even though I did it, it still doesn't work.....
Could you please suggest me something else?
Must you use Illumina Annotation? Can you try using any other annotation like Ensembl (Recommended) or Gencode? Section 2.4 of DEXSeq manual says that they have tested the script on Ensembl annotations and it may fail when used on other annotations.