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Question: BitSeq: Required input for 3' bias correction on transcripts.
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gravatar for Dave Gerrard
4.8 years ago by
Dave Gerrard40
Dave Gerrard40 wrote:
Hi Peter, Thanks for your reply. A lot of 'conventional' cDNA protocols created double-stranded DNA, from which the orginal strand information was lost but some RNA based library protocols retain the strandedness (see http://biology.stackexchange.com/questions/1958/difference-between- strand-specific-and-not-strand-specific-rna-seq-data). The difference would matter most when you have overlapping transcritps (common in small genomes) or you have no clue about transcription strand (ncRNAs with no poly-A). However, given that I am working on poly-A extracted RNAs from the sparse human genome, and BitSeq's mapping strategy, it's not too bad an assumption that the coding strand was the source. The reason I asked was because Cufflinks refuses to do non-uniform distribution without strand information (according to the manual). From you answer, it sound's like BitSeq infers the strand from the mapping direction (which is what I want) but does it then do something different for reads that map in the anti-sense direction? Dave On 27 January 2013 20:37, Peter Glaus <glaus@cs.man.ac.uk> wrote: > 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 > > 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/ > > > -- 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]]
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