Question: Rna-Seq, Cufflinks And Strand In Sam File
gravatar for Jimbo
7.4 years ago by
Jimbo120 wrote:

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

cufflinks rna bowtie • 3.1k views
ADD COMMENTlink written 7.4 years ago by Jimbo120
gravatar for Damian Kao
7.4 years ago by
Damian Kao15k
Damian Kao15k wrote:

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.

ADD COMMENTlink written 7.4 years ago by Damian Kao15k

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

ADD REPLYlink written 7.4 years ago by Jimbo120

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

ADD REPLYlink written 7.3 years ago by Deniz200
gravatar for Av_D
7.3 years ago by
Av_D20 wrote:

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.

ADD COMMENTlink written 7.3 years ago by Av_D20
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