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Pacific Biosciences of California, Inc. Message Board

  • paulieme60 paulieme60 Jul 5, 2013 3:25 PM Flag

    (HGAP), is based on only PacBio reads without the need for short-reads.

    Posted on July 5, 2013) De novo bacterial genome assembly: a solved problem?
    Pacific Biosciences published a paper earlier this year on an approach to sequence and assemble a bacterial genome leading to a near-finished, or finished genome. The approach, dubbed Hierarchical Genome Assembly Process (HGAP), is based on only PacBio reads without the need for short-reads. This is how it works:
    •generate a high-coverage dataset of the longest reads possible, aim for 60-100x in raw reads
    •pre-assembly: use the reads from the shorter part of the raw read length distribution, to error-correct the longest reads, set the cutoff in such a way so that the longest reads make up about 30x coverage
    •use the long, error-corrected reads in a suitable assembler, e.g. Celera, to produce contigs
    •map the raw PacBio reads back to the contigs to polish the final sequence (rather, recall the consensus using the raw reads as evidence) with the Quiver tool
    The approach is very well explained on this website. As an aside, the same principle can now be used with the PacBioToCA pipeline.
    In principle, this approach could result in a finished genome, i.e. a gapless contig per chromosomal element (chromosomes and plasmids). A more theoretical study confirms this:
    “Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library.” (Koren et al, arXiv:1304.3752)
    As always, the proof is in the pudding. There have been reports, and even a publication here and there, that the HGAp approach actually works. In this blog post I would like to add our experiences, which in short are that HGAP can indeed result in (close-to) finished genomes.
    (NEXT POST)

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    • Page 2 of 2 At the Norwegian Sequencing Centre,with which I am affiliated, we recently received several bacterial genome DNA samples for PacBio sequencing. Given our very positive first experiences with size selecting PacBio libraries using the BluePippin, see my previous post, we decided to use this instrument also for these samples. Four of the samples yielded very nice libraries, which were sequenced, two SMRTcells each, on our (recently upgraded) PacBio RSII instrument.

      For the Raw reads and full story, go to IHUB PACB M B for link

 
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