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RNA-Seq variants from 2-step alignments


Thank you for providing guidelines on RNA-Seq variant discovery. For our data, we are currently playing with multiple mapping methods and have noticed that 2-step alignments work "better" than 1-step alignments. By 2-step alignments, I mean using STAR as step1 and then take the unmapped from this and use another aligner (like Bowtie2) for alignment. If I use such a methodology, will there be an issue in variant calling when during splitting cigar strings I ask it convert the 255 MAPQ to another value (like 60 in the best practices example), since bowtie2 gives different MAPQ scores. Sorry if this seems like a stupid question, but I am just a little curious how such a thing might affect the variant calls. Any insights/comment on this will be greatly appreciated.



  • SheilaSheila Broad InstituteMember, Broadie, Moderator



    This should not cause any problems with reassigning mapping qualities, however, we cannot guarantee it because we have not tested it ourselves.

    Is there a reason you are not doing both alignment steps with STAR?


  • Is it ion torrent data? That kind of two-stepper seems to be commonplace for ion RNAseq data these days.

    The point of the second step is to align the initially non-mapping reads with more sensitive settings.

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