Bioscope With Cufflinks

Question

[Ramon Vidal]

Does any one here have ever used Bioscope with Cufflinks to do DE analysis? I am trying to use the BAM files generated by Bioscope to do it but all my output files have zero expression values (fpkm 0.0). I am using the recommended parameter "--library-type fr-secondstrand" and I also tried to convert the BAM to SAM and add the XS:A[+-] flag but it didn't work also. I don't know if it is the cause but I have two of these errors: "SAM error on line nnnnnnn: CIGAR op has zero length". Thank you

[Chris Penkett]

There is also a thread here on SeqAnswers with similar issues: http://seqanswers.com/forums/showthread.php?t=3551

I always used TopHat with SOLiD RNA-seq data and it worked to get alignments ready for CuffLinks.

Answer 1

[r.follador]

Doesn't directly answer your question, but I use following workflow to do DE analysis with SOLiD data:

• Do the alignment using LifeScope's Whole Transcriptome Mapping Analysis (I only need the BAM file)
• Do the gene count using HTSeq-count (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html)
• Do the DE analysis using DESeq2 (http://www.bioconductor.org/packages/2.12/bioc/html/DESeq2.html)

I very much prefer DESeq over Cufflinks; see for example Bullard et al., 2010 why the RPKM approach is not adivsable.