Can't use my SAM files for cuffdiff

[jasminegirl18]

Hi everyone,

I am running a cuffdiff with GTF files from cuffmerge and SAM files from my mapped reads (CLC Bio). However, I've got error notification that my SAM files are not aligned. I searched through the web and found this:

Do Cufflinks and Cuffdiff support both BAM and SAM?

Yes. If a SAM is supplied, a message will be output that the file is not a valid BAM file. However, Cufflinks will recognize this and treat the file as a SAM. When using a SAM file, you should include a proper header or ensure that the reads are lexicographically by chromosome and then numerically by left position. You can accomplish this sorting with the command sort -k3,3 -k4,4n in.sam > out.sam.

As I am running from Galaxy website and not using the LINUX setting, what should I do to keep my analysis going using cuffdiff. Thanks.

Jasmine

[yueluo]

Are your SAM files "sorted" by coordinates ?

[swbarnes2]

SAM files are far larger than .bam files, you are going to want to compress them for the long term, so since you are having problems with them now, you should probably just compress them already.

[jasminegirl18]

@yueluo May I know how can I do that? Does that means that I assigned coordinates to each mapped sample (different conditions) so that cuffmerge can recognise it? Which tool can I use in Galaxy?

Hope to get reply from you soon. I am really foreign to bioinformatics tools. Thanks guys =)

[yueluo]

What did your error message look like?
Here is a quote from cufflinks on how to sort the sam file:

The SAM file supplied to Cufflinks must be sorted by reference position. If you aligned your reads with TopHat, your alignments will be properly sorted already. If you used another tool, you may want to make sure they are properly sorted as follows:

sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted

[jasminegirl18]

@yueluo Thanks. I do realise it now that it will be easier for me to align my reads using Tophat instead of CLC Bio. The problem is because I am using cufflink thru Galaxy website and I am unsure how to key in the command stated above. If I am not wrong, the command stated above is only LINUX based right?

Thanks for your help =)

[jasminegirl18]

@yueluo By the way, are you a frequent user of galaxy?

[yueluo]

@jasminegirl
Sorry,no... I have access to local clusters/servers.

[jasminegirl18]

@yueluo Oh i see. But thanks anyway for your help. I will update you on my progress. Thanks.