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  • RGLADSTONE
    Junior Member
    • Aug 2011
    • 1

    #1

    Miseq assemblies

    Bacteria sequencing. Nextera XT library prep. Triplicate technical replicates from a single MiSeq run 2 x 250 PE, de novo assembly on MiSeq gives ~450 contigs for all three. Optimized velvet assembly from fastq's gives 200 contigs for two samples and 800 contigs for one. Why would there be so much of a difference between 3 technical replicates for one method of assembly and not for the other?
    To make things more complicated the triplicate samples are from a single strain but three individual DNA extractions.
    Last edited by RGLADSTONE; 01-11-2013, 02:28 AM.
  • mwatson
    Member
    • Aug 2010
    • 13

    #2
    Originally posted by RGLADSTONE View Post
    Bacteria sequencing. Nextera XT library prep. Triplicate technical replicates from a single MiSeq run 2 x 250 PE, de novo assembly on MiSeq gives ~450 contigs for all three. Optimized velvet assembly from fastq's gives 200 contigs for two samples and 800 contigs for one. Why would there be so much of a difference between 3 technical replicates for one method of assembly and not for the other?
    To make things more complicated the triplicate samples are from a single strain but three individual DNA extractions.
    1) is there a reference? If so, map and then estimate the insert size
    2) Try FLASH to see if the reads overlap
    3) Trim to 100bp PE and assemble again -> does this help?
    4) Randomly choose 10%, 25%, 50% of the data, assemble again, does this help?
    5) have you checked for adapters?

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