Cufflinks- found spliced alignment without XS attribute

Question

[Lindsay44]

I am getting the following command line message (various times) when running Cufflinks:

BAM record error: found spliced alignment without XS attribute

What does this mean? Is the program running correctly? Is the output reliable?

Answer 1

[michael.ante]

Hi Lindsay44,

it means that your aligner did not report this optional attribute to the alignments. The alignments themselves can be correct and valid, but Cufflinks need this optional attribute. If you have a strand-specific library, you can circumvent this issue by providing Cufflinks the library-type parameter.

Otherwise, you'll need to re-run the alignment with a program which reports the XS attribute (e.g. Tophat2, STAR with the "--outSAMstrandField intronMotif" parameter, bbmap with the xstag setting...).

Cheers,

Michael

[Lindsay44]

Okay thank you.

Is there an option for this in the HiSat2 aligner?

How will my results be affected if I don't make this change?

[michael.ante]

According to the HiSat2 Manual:

We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000".

I'd give STAR or BBMAP a try.

[Lindsay44]

It is a little late for me to switch at this point. If I don't change anything, will the results be inaccurate?

I have single end data- how should this be put into Hisat2?