[mira_talk] Collecting DNA from bacterial cultures, was: suggestion for sequencing companies

  • From: Peter Cock <p.j.a.cock@xxxxxxxxxxxxxx>
  • To: mira_talk@xxxxxxxxxxxxx
  • Date: Thu, 12 Apr 2012 10:07:58 +0100

On Tue, Apr 10, 2012 at 1:37 AM, Bastien Chevreux <bach@xxxxxxxxxxxx> wrote:
> Detour - Sequencing -
>
> For me, every "sequencing project", be it genomic or transcriptomic, really
> consists of four major phases:
>
> data generation
> This can be broadly seen as everything to get the DNA/RNA ready to be sent
> off to sequencing (usually something the client does), the library prep at
> the sequencing provider and finally the sequencing itself (including base
> calling). An area of thousand pitfalls where each step (and the
> communication) is crucial and even one slight inadvertence can make the
> difference between a "simple" project and a "hard" project. E.g.: taking DNA
> from growing cells (especially bacteria in exponential growing phase) might
> not be a good idea ... it makes assembly more difficult. Some DNA extraction
> methods generate more junk than good fragments etc.pp
> The reason I am emphasizing this is simple: nowadays, the "sequencing"
> itself is not the most expensive part of a sequencing project, the next two
> steps are (most of the time anyway).
> assembly & finishing
> Still a hard problem....

Hi Bastien,

We're likely to be sequencing a few more bacteria soon, and your comment
about avoiding collecting DNA from the exponential growth phase intrigued
me. Does this essentially introduce coverage bias towards the origin of
replication (and therefore makes things harder to assemble)? i.e. there will
be more copies of the DNA in that part of a circular genome because some
will be mid duplication?

I don't recall coming across this advice before - if you have any references
that would be educational. Or do you have a specific recommendation
instead when dealing with a bacterial culture - e.g. allow them to reach
saturation, or put them on ice before harvesting DNA?

Thanks,

Peter

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