I have RNAseq data generated from poly-A capture RNAs. There appears to be a strong 3' bias to the mapped reads but the library prep was NOT strand specifc. I would like to correct for the positional bias in the mapped reads and Bitseq has a 'uniform' parameter which can be set to 'FALSE' if reads are not uniform. Please can you tell me if this requires 'stranded' mapped read information (as per the cufflinks manual) or whether It will work without knowing the source strand? Thanks, Dave Gerrard -- david.gerrard@manchester.ac.uk Bioinformatics | The University of Manchester | Michael Smith Building | Room B.1079 | Oxford Road | Manchester | M13 9PT T: 0161 275 5737 http://personalpages.manchester.ac.uk/staff/David.Gerrard/ [[alternative HTML version deleted]]

Hi Dave, as far as I understand the RNA-seq protocols, the "endedness" of reads is determined depending on whether they were generated in first-strand synthesis or second-strand synthesis. Either way, this can be also determined based on transcripts "strandedness" (in our case known from annotation) and read being aligned to sense or anti-sense strand of the transcript. So BitSeq tries to determine end for each read and it is safe to use the non-uniform model. If you want to make sure that everything runs OK you can use verbose flag to get more information and check the number of reads that are reported in the pre-processing part and watch for any unexpected warnings. Regards, Peter. On 01/25/2013 12:02 PM, Dave Gerrard wrote: > I have RNAseq data generated from poly-A capture RNAs. There appears > to be a strong 3' bias to the mapped reads but the library prep was > NOT strand specifc. I would like to correct for the positional bias in > the mapped reads and Bitseq has a 'uniform' parameter which can be > set to 'FALSE' if reads are not uniform. Please can you tell me if > this requires 'stranded' mapped read information (as per the cufflinks > manual) or whether It will work without knowing the source strand? > > Thanks, > Dave Gerrard > -- > david.gerrard@manchester.ac.uk <mailto:david.gerrard@manchester.ac.uk> > > Bioinformatics | The University of Manchester | Michael Smith Building > | Room B.1079 | Oxford Road | Manchester | M13 9PT > T: 0161 275 5737 > > http://personalpages.manchester.ac.uk/staff/David.Gerrard/ [[alternative HTML version deleted]]