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Old 05-29-2013, 10:24 AM   #21
sdriscoll
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Also I don't want to send you down a confusing path. I don't mind providing you with some help to get that pipeline working. One more thing to consider - do you expect insertions/deletions to be important? If so then RSEM may not be what you want since it uses bowtie1 for alignments. eXpress is a similar solution and with eXpress you can use alignments from bowtie1, bowtie2, bwa (with some tweaking) and really any aligner that can output all possible alignments for a given read. These tools attempt to disambiguate the alignments to a set of gene/protein/transcript sequences giving you "unique" mappings for even reads that can align equally well to several references. I've done a bit of benchmarking and honestly I haven't seen great results from eXpress but RSEM does pretty well. Both work VERY well if you are able to sum counts of sequences together for sequences that share exons or share sequence (as in multi-copy genes or alternatively spliced genes). They work OK in terms of per-sequence level counts - certainly better than what the aligners can do on their own - but certainly not perfect. Just keep in mind that you're per-sequence expressions will likely contain some false positives (maybe a lot...) and will also likely be missing a few true positives. In the end you're knowledge of which sequences in your database share sequence or share exons will help you immensely in getting stable and reliable read counts.
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Old 05-31-2013, 05:46 AM   #22
bob-loblaw
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Quote:
Originally Posted by sdriscoll View Post
Also I don't want to send you down a confusing path. I don't mind providing you with some help to get that pipeline working. One more thing to consider - do you expect insertions/deletions to be important? If so then RSEM may not be what you want since it uses bowtie1 for alignments. eXpress is a similar solution and with eXpress you can use alignments from bowtie1, bowtie2, bwa (with some tweaking) and really any aligner that can output all possible alignments for a given read. These tools attempt to disambiguate the alignments to a set of gene/protein/transcript sequences giving you "unique" mappings for even reads that can align equally well to several references. I've done a bit of benchmarking and honestly I haven't seen great results from eXpress but RSEM does pretty well. Both work VERY well if you are able to sum counts of sequences together for sequences that share exons or share sequence (as in multi-copy genes or alternatively spliced genes). They work OK in terms of per-sequence level counts - certainly better than what the aligners can do on their own - but certainly not perfect. Just keep in mind that you're per-sequence expressions will likely contain some false positives (maybe a lot...) and will also likely be missing a few true positives. In the end you're knowledge of which sequences in your database share sequence or share exons will help you immensely in getting stable and reliable read counts.
Well alternatively spliced genes won't be a problem, it's all bacteria I'm mapping to. RSEM I have been playing around with, but I have a pretty large sample size, and realigning all of them would be very time consuming, obviously I'll do it if necessary but I'd prefer not to have to. I haven't tried eXpress yet, but I will.

When I was mapping with bowtie2 I left it's reporting mode in default (i.e. report only the best alignment) but eXpress wants to be able to select the best alignment itself. Do you think this will be a big issue?
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