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  • one transcript many genes "chimeric"

    The problem:
    I am trying to assemble the transcriptome of Belgica the antarctic midge. Some of our assembled transcripts are much larger than they should be, and they contain multiple genes . In the ones that I have dug into in depth, these genes are adjacent on the chromosomes of the draft genome. We have no reason to believe that these are biological in origin.

    The situation:
    I am working with three lanes of RNA-Seq data from a solexa machine. We have about 35 million paired end reads (70M total). Each read is 76 bp. At the same time we have a genome assembly project that is not under my direct control. It is against this draft genome that I am attempting to assemble the transcriptome with tophat/cufflinks.

    Has anybody run into this before. I have been pulling my hair out trying to tweak the input parameters of tophat and cufflinks in order to eliminate this. I have also tried filtering my reads. I accepted sequences where 75% of the sequence had a phred score of 38 or better. Both mates had to pass in order to be included.

    I have found one or two threads on various forums and they were not very helpful. The most helpful idea anyone had was to contact the authors. I tried that, but I am not holding my breath. Their automated message (below) openly stated that they may not contact me back. nice. I really appreciate any help you guy and gals can give me.

    Dear Tophat/Cufflinks User,

    Your message has been received and will be forwarded to the appropriate project members. Due to the large numbers of e-mails we receive, a response may not be immediate. We focus first on high priority bug reports before answering general questions and sometimes do not respond to repeat bug reports when a fix is already in the works. In the meantime, please have a look at the links below, which may aid in answering your questions.

    TopHat Manual: http://tophat.cbcb.umd.edu/
    Cufflinks Manual: http://cufflinks.cbcb.umd.edu/
    SeqAnswers Forum: http://seqanswers.com/

    Regards,

    The TopHat and Cufflinks Teams
    Last edited by BugSeq; 02-13-2012, 09:32 AM. Reason: forgot to add the Cufflinks letter

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