Cuffdiff doesn't accepted sorted bam files that worked in cufflinks

Question

[lzswindon]

I have successfully run through cufflinks and cuffmerge on my STAR aligned data, but am running into an error on cuffdiff:

cuffdiff merged.asm/merged.gtf \
H_400Pa_2079out/Aligned.out.sorted.bam.bam, H_400Pa_2085out/Aligned.out.sorted.bam.bam \
H_60kPa_2080out/Aligned.out.sorted.bam.bam, H_60kPa_2086out/Aligned.out.sorted.bam.bam

This error repeatedly appears on the screen:

BAM record error: found spliced alignment without XS attribute

I had encountered this readout when running cufflinks, but addressed it by using samtools sort on my original bam files. I'm not sure how to address this new occurrence, since my bam files are already sorted. Help would be appreciated! Thanks.

Answer 1

[lzswindon]

Ah, silly me, I left out -library-type fr-firststrand in my cuffdiff command, which I'd used in cufflinks. So full command should be:

cuffdiff -library-type fr-firststrand merged.asm/merged.gtf \
H_400Pa_2079out/Aligned.out.sorted.bam.bam, H_400Pa_2085out/Aligned.out.sorted.bam.bam \
H_60kPa_2080out/Aligned.out.sorted.bam.bam, H_60kPa_2086out/Aligned.out.sorted.bam.bam

[Ghazala.S]

Thanks for the update. I had the same error and got resolved once I added the library type.