Hey,
It's my first time to use HISAT2 for alignment and the manual is full of parameters that got me confused.
I need to write down a command that would include the following:
map against hg19, my samples are paired-end, I need to have xs attributes to the output, and I need the output to be compatible with Stringtie for downstream analysis.
Have anyone worked on this pipeline before and can help me?
Thank you!
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dina.hesham139
hisat2 -x /path/to/hg19/indices -1 sample_1.fq.gz -2 sample_2.fq.gz | samtools view -Sbo sample.bam -
The resulting BAM file should work with stringTie or cufflinks. You probably want the --known-splicesite-infile option though.
Edit: You probably need to sort and index the BAM file. At least cufflinks would require that.
Hi Devon,
I work with dog genome. If I don't have premade indexes such as genome_tran or genome_snp_tran, should I go ahead to put my gtf file in the --known-splicesite-infile option or should I no using any gtf at all?
Thanks!
I think the GTF needs to be preprocessed first (there's a script for that that comes with hisat2), but aside from that yes.
Thanks for your suggestion! I will process it first with extract_splice_sites.py.
Hi Devon, could you tell me what is the meaning of the '-' symbol at the end of your samtools view command above (after 'sample.bam')? I am running a similar code without the '-', and sometimes it works fine but other times the program ends with a parsing error.. thanks!
- means "read from the pipe (|)" in this case. This is particular to samtools. If you're running samtools on a file then you would never use -.
You'll likely get slightly better alignments by using that and therefore slightly higher counts for DE testing.
The defaults should be fine. The only other thing to change is the number of threads used.
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For the
--known-splicesite-infile, it was stated in the manual:Is using genome_tran index would substitute the genome+ the annotation gtf file parameters in Tophat?
Yeah, if you want to download the prebuilt indices then just get genome_tran and call it done.
Thank you!!!
One more question, for alignment, what would you generally recommend STAR or HISAT2?
I generally prefer STAR, though it requires significantly more RAM.