Cuffquant errors after using HISAT2

Question

[ilikebing2000]

Hi,

I have 2 sequence reads archive and I want to do some RNA-Seq analysis on them.

Archive 1 : SRR1177960 (FastQ Files : SRR1177960_1, SRR1177960_2)

Archive 2 : SRR1177961 (FastQ Files : SRR1177961_1, SRR1177961_2)

I aligned both archives using HISAT2, and I got the results (BAM files).

Then I pass the results of each archive separately to Cufflinks, and I got the results (GTF files).

Then I pass the results to Cuffmerge, and I got the result (GTF file).

Then I pass the Cuffmerge result + HISAT2 result of archive 1 to the Cuffquant, and I got the result (CXB file).

BUT when I pass the Cuffmerge result + HISAT2 result of archive 2 to the Cuffquant, I got error

BAM record error: found spliced alignment without XS attribute

I attached the full text of error : cuffquant_error_archive2.txt

Can you help me?

(I forgot to say that I'm using Galaxy usegalaxy.org))

Thanks.

Answer 1

[ablanchetcohen]

Just set the "Penalty for non-canonical splice sites" to an extremely high value.

HISAT (I didn't include the smaller and greater then symbols in the citation below, because Biostars then messes up the presentation. It should be XS:A:greater then A smaller then)

XS:A:A Values of + and - indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie. We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000".

HISAT2 (not available yet on usegalaxy.org)

--dta-cufflinks

Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment.

[ilikebing2000]

Sorry for late reply, HISAT2 is available on UseGalaxy.org, It uses HISAT Version 2.0.3. As you said, I use the --dta-cufflinks option (Report alignments tailored specifically for Cufflinks), and the problem solved! Thanks a lot!