How many multiple alignments to allow?

hlwright · 04-14-2011, 12:40 AM

Hi everyone, I am working with human transcriptome sequenced by SOLiD - at the moment single read, but plan to try mate paired next - and using Bowtie/TopHat/Cufflinks to align reads. I am fairly new here and I have a simple but very important question.

How many multiple alignments should you allow for each read?

Its a difficult question when a read will map equally well to more than one location (how do you call which is best?). As I understand it TopHat, as default, reports up to 40 different alignments per read. I know this can be adjusted using the -max-multihits option, but this gets rid of every alignment for reads mapping at multiple sites. Bowtie has options for --best --strata but this still reports all reads which are best (at least when I run the command it does). There are also options for -k, -a, -m, -M.

How am I supposed to know or choose which option(s) to apply to my data? Surely if I allow the software to report all possible mappings then I am biasing my data for later stages when RPKM is calculated?

I just wondered what the wider community was doing about this, and what sort of parameters you are applying to your data?

Many thanks
Helen

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04-14-2011, 12:40 AM	#1
hlwright Member Location: Liverpool, UK Join Date: Feb 2011 Posts: 30	How many multiple alignments to allow? Hi everyone, I am working with human transcriptome sequenced by SOLiD - at the moment single read, but plan to try mate paired next - and using Bowtie/TopHat/Cufflinks to align reads. I am fairly new here and I have a simple but very important question. How many multiple alignments should you allow for each read? Its a difficult question when a read will map equally well to more than one location (how do you call which is best?). As I understand it TopHat, as default, reports up to 40 different alignments per read. I know this can be adjusted using the -max-multihits option, but this gets rid of every alignment for reads mapping at multiple sites. Bowtie has options for --best --strata but this still reports all reads which are best (at least when I run the command it does). There are also options for -k, -a, -m, -M. How am I supposed to know or choose which option(s) to apply to my data? Surely if I allow the software to report all possible mappings then I am biasing my data for later stages when RPKM is calculated? I just wondered what the wider community was doing about this, and what sort of parameters you are applying to your data? Many thanks Helen