I've got some doubts on the hisat2
--rna-strandness option and its output for downstream analysis. Is it expected to see a difference in alignment and counting results given the default usage of
--rna-strandness in hisat2 followed by
htseq-count -s reverse on a strand-specific assay?
Please see below.
I understand that the
--rna-strandness option produces an XS tag to indicate where a transcript is from (on the + or - strand) for downstream transcriptome assembly analysis. I have a paired-end stranded sequencing library that was aligned to the genome using hisat2 without specifying the
--rna-strandness (in other words, the default unstranded was the usage). Following this, the reads were assigned to genes using htseq-count and this time
-s reverse was specified given the strand-specific sequencing assay type.
--rna-strandness is for transcriptome assembly using the XS tags generated and htseq does not use XS tags for counting, I presume there should be no practical impact from the above. Could you also shed light on this? in case I may have been overlooking other facts of the usages of the tools.
To help verify the above, I re-aligned and counted the reads from 2 samples by switching on
--rna-strandness RF in hisat2. I attach the alignment and count features info. below for assessment.
Overall alignment rate of Sample 1:
94.52% (--rna-strandness RF) 94.12% (--rna-strandness unstranded)
Overall alignment rate of Sample 2:
94.57% (--rna-strandness RF) 94.15% (--rna-strandness unstranded)
Feature counts of Sample 1 (following --rna-strandness RF + -s reverse):
__no_feature 6327294 __ambiguous 2954776 __too_low_aQual 3784481 __not_aligned 688856 __alignment_not_unique 4858182
Feature counts of Sample 1 (following --rna-strandness unstranded + -s reverse):
__no_feature 6291151 __ambiguous 2911298 __too_low_aQual 4075017 __not_aligned 754400 __alignment_not_unique 16136045
Feature counts of Sample 2 (following --rna-strandness RF + -s reverse):
__no_feature 5417882 __ambiguous 1708510 __too_low_aQual 3532352 __not_aligned 564596 __alignment_not_unique 2859501
Feature counts of Sample 2 (following --rna-strandness unstranded + -s reverse):
__no_feature 5359434 __ambiguous 1676091 __too_low_aQual 3813344 __not_aligned 623122 __alignment_not_unique 2891792
These results look comparable to me across pipelines.