Rna-Seq, Cufflinks And Strand In Sam File

Question

[Jimbo]

Hi, I have some Illumina data, RNA-seq, 34 bp long reads, unpaired, no strand information.

I have aligned to the reference genome using bowtie, and analysed the results using DESeq quite successfully.

I want to use cufflinks to try to find areas outside of known genes that are expressed.

Do I need to go through the SAM file and change the "strandedness" column to "*" ? Bowtie gave my data a strand value, but really this means nothing, the reads could have come from either strand, it's just the way they were sequenced as I understand.

Will this affect my cufflinks results? They talk about an XS tag with spliced alignments in the manual but I don't quite get it.

Thanks in advance

Answer 1

[Damian Kao]

Cufflinks has a --library-type option which will account for strandness/unstrandness depending on your library. Default setting is unstranded, so you should be fine running on default settings.

[Jimbo]

Perfect, thanks, that's what I thought, it's because the example had a "*" in that position so I thought I'd check.

[Deniz]

Hello Jimbo, I also used bowtie for alignment for single paired. my command is like that -S -q -p 8 -a --best -v 2 -m 1 --strata REferans file.txt

I will use this alignment for SNP calling. I allow 2 mismatching is there anything wrong with my command?

Could you share your bowtie command ? Thanks

Answer 2

[Av_D]

the auxiliary 'XS' tag should have a value for all spliced alignment records (TopHat support this). If you are running Bowtie alone, you may not have this 'XS' in your SAM/BAM.