hisat2 run gave 0% alignment

Question

[vinayjrao]

Hi,

I analyzed some RNA-Seq data using the old Tuxedo pipeline (Tophat, Cufflinks and cimmeRbund), but learned that there is an improvisation to the tuxedo protocol, so I ran hisat2, but am constantly getting 0% alignment. Is there anything that I'm doing wrong? I'm posting my command below -

hisat2 -p 8 --dta -x ../../data/ref/hg38 -1 ../../data/fastq/BT474_38_1.fastq -2 ../../data/fastq/BT474_38_2.fastq -S ../../data/hisat/bt474/bt474.sam

This was the command I ran. I would also like to point out that hisat2-build -p 8 -c ../../data/ref/hg38.fa ../../data/ref/hg38 took less than 5 seconds to run. Is that normal?

Thanks.

Answer 1

[Satyajeet Khare]

Change

hisat2-build -p 8 -c ../../data/ref/hg38.fa ../../data/ref/hg38

to

hisat2-build -p 8 ../../data/ref/hg38.fa ../../data/ref/hg38

[vinayjrao]

Thanks a lot, Satya. Could you please explain to me why this works though?

[Satyajeet Khare]

If I am not wrong you will have to provide actual sequence in the cmd line with -c option. According the manual...

-c                      reference sequences given on cmd line (as
                            <reference_in>)

[vinayjrao]

Oh, OK. Thanks again. I didn't think of that.