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  • 454 expected sequences and Tag for amplicon sequencing

    Hi everybody,
    We plan to do a complete run with 454 sequencing (100mb). I already did a 16th of a run (GS FLX 250pb average) and it has generated only 6000 sequences (I was expected 12000) the compagny I worked with to do the 454 said that it is normal and if we want to do a complete run we will have only 300000 sequences instead of the 400000. I did amplicon sequencing with 9 samples pooled together (with 9 different 4bp tags between the adaptator and the primer). Now I have been in touch with others compagny and they said that I should have 400000 sequences and even more (one compagny reckon that they can achieve 30% more). This compagny suggest that the low nb of sequences from the previous 16th of run may be due to a bad design of the tags (redundancy) and I have effectively some that are redundant but it is not the sample that have the less sequences.
    Did anyone run a complete run and can we really have the 400000 sequences promised by roche with GS FLX (250pb read average). Does the design of the tag can really be a problem (I thought 454 was precise at 99.9%).

    Thank you for answering.

  • #2
    We did a complete run and got 462074 reads with average 214 bp after trimming the adaptors.

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    • #3
      This paper: http://www.ncbi.nlm.nih.gov/pubmed/18264105

      ...reports 437,544 reads of 240-280bp after trimming and removal of low quality reads.

      It's also a useful paper for amplicon sequencing with tagging/indexing...

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      • #4
        Genome Viewer

        Originally posted by platyias View Post
        We did a complete run and got 462074 reads with average 214 bp after trimming the adaptors.
        How did you visualize everything after assembling and trimming.
        We have whole genome data that EagleView is unable to show with annotations (its limited to 10MBP). Someone suggested Gbrowse in the Bioinformatics thread, but I was hoping for a more readily available solution ... anyone?

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