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  • nbahlis
    replied
    Tophat and Proton

    Did anyone figure out the type of library to use with Ion Torrrent Proton reads when using tophat? --library-type specified as either fr-firststrand or fr-secondstrand?
    Also has anyone used tophat-fusion-post with Proton reads? I always get 0 fusions in the post directory!

    Leave a comment:


  • qserenali
    replied
    Tophat --library-type option

    I have Ion Proton single end/strandness RNA-Seq data and have been trying to figure out what options to give to --library-type option. I used the same dataset (~413982 reads) with no --library-type specified or with --library-type specified as either fr-firststrand or fr-secondstrand using the hints from Tophat FAQ page, but the counts from junctions.bed are quite comparable. I wonder whether this is because the strand info is only applicable to paired-end data (as some of the posts suggested)? For single end data, could tophat leverage strand info?

    #I am not sure which library type to use (fr-firststrand or fr-secondstrand), what should I do?
    #
    #One possible way to figure out the correct library-type is to run TopHat with a small subset of the reads (e.g., 1M) as follows.
    #run TopHat with fr-firststrand and count the number of junctions in junctions.bed (one of the output files from TopHat)
    #run TopHat with fr-secondstrand and count the number of junctions in junctions.bed
    #Since the splice junction finding algorithm of TopHat makes use of library-type information (if provided), one of the two TopHat runs would result in many m
    ore splice junctions than the other one. You can then use the library type that gives more junctions. If this is not the case TopHat might not work well with
    your sequencing protocol. Please let us know more details about your protocol so we can add support for new library types.

    > more tophat_coverage-search_no_library-type_option/junctions.bed|wc -l
    188
    > more tophat_no-coverage-search_fr-secondstrand/junctions.bed|wc -l
    181
    > more tophat_no-coverage-search_fr-firststrand/junctions.bed|wc -l
    175

    I would also like to confirm if tophat assumes fr-unstranded when no --library-type option is provided? While both tophat manual and tophat -h mention three supported library-type options, cufflinks manual mentioned the same library-type options and cufflinks -h on the other hand mentioned 7 supported options below. I wonder whether exactly how many options are there and whether the library-type only applies to paired-end data?
    Supported library types:
    ff-firststrand
    ff-secondstrand
    ff-unstranded
    fr-firststrand
    fr-secondstrand
    fr-unstranded (default)
    transfrags

    I am using TopHat v2.0.8 and cufflinks v2.1.1.

    Leave a comment:


  • vinay052003
    replied
    Thanks a lot for the useful info.

    Leave a comment:


  • Wei-HD
    replied
    http://seqanswers.com/forums/showpos...95&postcount=9

    Originally posted by jingerlu View Post
    I have the same question........

    Leave a comment:


  • jingerlu
    replied
    I have the same question........

    Leave a comment:


  • Wei-HD
    started a topic Tophat --library-type option

    Tophat --library-type option

    Dear all,

    I have a quick question about the "--library-type" option in the latest Tophat version 1.2.0:

    If the reads are from single end, strand non specific library preparation, then one should use "--library-type fr-unstranded", but the description about this standard Illumina type sounds like only for paired-end reads.

    So Tophat will treat the reads as strand specific by default if one does not specify *--library-type* option. Does anyone have experience about this?

    Many thanks!

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