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I am going to get some data from plasmid sequencing to identify SNPs on the plasmids. What is done in the lab is the following:

  • The plasmids are purified by size.

  • We amplify the plasmids using the phi29 polymerase. The polymerase will go through the plasmid multiple times. Hence we get the same sequence concatenated multiple times. My question is related to this step:

  • Finally, We sequence it using Oxford Nanopore.

My question is:

On step two, I wrote that "we get the same sequence concatenated multiple times". For me, this a potential source of information to correct the base calls prior to aligning them to the reference. Since you have the same sequence multiple times (concatemers).

What I would like to know is if there is a tool that uses the information of the concatemers to improve the base calls. I have tried to find some methods but found none. I know that PacBio has a similar flavour using the "circular consensus calling", but I have not found any methodological explanation.

thanks!

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  • $\begingroup$ Nice project! Do you know the length of the (linearized) plasmids? I haven't heard of any tool similar to this but I don't usually analyze long-reads sequencing data. Good luck with your project $\endgroup$
    – llrs
    Nov 8, 2018 at 10:22
  • $\begingroup$ I don't, the project aim is to detect which plasmids a bacteria contains. Thanks for your answer $\endgroup$
    – Praderas
    Nov 8, 2018 at 11:26
  • $\begingroup$ Then the project is more complicated, because if you don't know the length of the repetitions of the plasmid you'll need to search for a range of size of the repetitions, which can be longer/more difficult (depending on how you compare the different portions of the reads) $\endgroup$
    – llrs
    Nov 8, 2018 at 11:48

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Yes, there is a tool to do this called R2C2 by the Vollmers lab at UC Santa Cruz.

https://www.biorxiv.org/content/early/2018/06/04/338020

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  • $\begingroup$ That was definetively the tool I was looking for. Thank you very much! $\endgroup$
    – Praderas
    Nov 8, 2018 at 18:41

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