hello all,
I'm using STAR to 2-pass alignment and htseq-count to output gene count table. But my htseq-count result table is a bit strange, like follow:
__no_feature 5802774
__ambiguous 4365152
__too_low_aQual 0
__not_aligned 0
__alignment_not_unique 0
all alignments are in no feature or ambiguous couters. Why does this happen. My code is:
htseq-count -s no Aligned.out.sorted.sam gencode.v28.annotation.gtf > samp.htseq-count.tab
Any one has got same problem?
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lilingjoyo •
-s no
This is (imho) the problem. Perhaps your library is stranded. I'm saying this because I see ~ half of the scores in __no_feature and another half in __ambiguous. Try -s yes!
In fact, I tried both. They put almost same result. All reads are in "no feature" or "ambiguous" group.
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Does the gtf used for htseq-count contain the same chromosome identifiers as your alignment in sam format?
I thinks so. In my chrName.txt one of my star index files, the chromosome id is in the form of chr#. In my gencode gtf file, it's same.
To save your time and shorten the pipeline, you can use STAR's
--quantModeoption (have a look in manual). It will generate exactly the same read-counts as htseq-count (note that probably this would not solve the issue you posted here).