9 Responses

  1. flxlex
    flxlex
    February 27, 2014 at 2:16 pm |

    Thanks, Nick! And congratulations on your results…

    Two comments:

    - your aside on Oxford read lengths is entirely correct: they will get much better lengths once they adjust the fragmentation and library prep conditions
    – you could (should) run Quiver multiple times on the assembly, to get rid of the last indels

    Lex

  2. scbaker
    scbaker
    February 27, 2014 at 4:43 pm |

    Great post, Nick! This will be really helpful for those getting into PacBio sequencing. In terms of finding the right provider, I’d humbly recommend checking out our service – AllSeq’s Sequencing Marketplace http://allseq.com/information/need-sequencing

    We’re adding PacBio providers all the time and we can help people find the best one for their project (whether it be price, turn around time, or application expertise).

    Shawn

  3. Lutz
    Lutz
    February 28, 2014 at 8:16 pm |

    Hi,
    what are your experiences with the seeming DNA-amount-dependency of the Blue Pippin size cut-off; would you have explanations for it?

    We did generate a seemingly nice library with the bulk of fragments ranging in size from 15 to 25 kb (according to a CHEF gel); it had however a low concentration (we loaded 1 ug on the Blue Pippin). We ran it unfortunately with an older BluePippin protocol and a size cutoff of 10 kb and only recovered 28 ng in total (on the AB you can see some traces of the library starting from 10 kb).
    We were told that the Blue Pippin is DNA amount sensitive and that we should use the 4 kb cutoff as in your table. With a second library (same size range) we did load 1.8 ug on the BP and selected the 4 kb cutoff. We recovered 650 ng in total and the size cutoff visible on the BA trace seems correct at 4 kb. Since the BA traces before and after BP otherwise resemble each other I suspect we should not have followed the recommendations and should have used a higher cutoff (at ealst 7 kb?).
    Is the DNA migration changing at low DNA concentrations? – Is simply a certain amount of DNA always getting stuck in the apparatus (but why then change the cutoff?)?

    Nick, which chemistry did you end up using for your data?

  4. Cliff Beall
    Cliff Beall
    March 3, 2014 at 4:23 pm |

    Thanks for the article. I have been struggling with whether to use PacBio, but am worried about the cost, especially for numbers of genomes. Do you have any thoughts on combining PacBio and Illumina data?

  5. Lutz
    Lutz
    March 14, 2014 at 5:57 am |

    Hi Nick,

    thanks for the offer. We will do some more experiments and see if there are inconsistencies or if there is alchemy involved.

  6. paolaflorez
    paolaflorez
    May 16, 2014 at 8:52 am |

    Hi Nick, thanks for the blog. PAcBio sequencing of bacterial genomes is very interesting to me, since our institute just purchase a RSII and it is sitting on our floor. My job to to sequence as much as I can now and get people interested as well as getting the beast on-line in a production mode for the institute. I have something very similar to what you are seeing a Singapore strep. pneumo. isolate, which down to 2 unitigs (~2million bp and ~89k bb). The coverage for both unitigs is about 86x. This leads to be believe the smaller one is not a plasmid, plus the smaller one is a bit to big for a plasmid. I am happy with the information at hand and am currently digging into attempting to compare the PacBio assembly to the Illumina assembly and making some progress. I had built a 10kB library, since that was part of the training run for the machine. Next week I will build a 20kB library, but may I ask what have you done to try to put your 2 unitigs together? Maybe I’m missing something.
    Best,
    Paola

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