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Large No. small fragments Deep Amplicon Seq (454-GS-FLX)-demultiplexing problems



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  • Large No. small fragments Deep Amplicon Seq (454-GS-FLX)-demultiplexing problems

    Hello everyone,

    I just got the results of my Deep Amplicon sequencing project (fusion primer) from my sequencing service provider (platform used: Roche GS-FLX). As you can see below (***), the results are not positive, and they have not explained the principal reasons (experimental) why this has happened (twice-they repeated the run); however, they are still asking for the big budget located for this sequencing service.

    ***Services provider message: ***

    The total number of reads produced for the run was 214,647 post Amplicon Analysis.

    ___________________No. sequences // No. bases (bp) // Average length // GC (%)
    Total demultiplexed __// 27502 //__ 9678243 // _______ 349 // ___ 59.04
    Total bin __________ // 214647 // __ 26545798 // ______ 123 // ___ 57.18

    The results are very similar to your first ¼ PTP of sequencing which produced 26,363 reads. Unfortunately, due to the large number of small fragments in the sequencing run it has not been possible to demultiplex all the results. It’s likely that there’s something unique about the sequence of your samples which are resulting in the short read lengths that we are seeing. We are currently investigating to see if we can discover the cause of the short fragments as it’s not something we have seen before (they said the same two months ago, and I haven’t got an clear explanation yet).


    Additional information about the DNA targeted regions: I am trying to sequence multiple exons from the several duplicated genes from the MHC from different close related species. The sequences of primers were designed according to DNA sequences previously obtained for the group of species we have been studying. The process was performed completely by my service provider (from primers’ synthesis to 454 sequencing).

    More than the money (which is still important for our research), I am interested in figure out which are the possible reasons that can explain why this has happened. I already read the thread cited below, but I think those answers apply more to my sequencing service provider:
    http://seqanswers.com/forums/showthread.php?t=15838 (I think those are standardising alternatives). Additionally, I want to know if manually de-multiplexing can be a solution (I mean, my own custom perl script)? if so, Any special suggestion to prevent the lost of valuable information?

    On the other hand, should I ask to my provider to guarantee the service they provided, or in the reality, my sequences can be the real problem?

    Many thanks, and sorry for the long post and multiple questions.

    Best regards,

    Last edited by aperdomos; 09-17-2012, 10:44 PM.

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