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

On Apr 12, 2012, at 11:07 , Peter Cock wrote:
> 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?

Hi Peter,

yes, and yes. I do not remember whether I've shown corresponding slides during 
my talk in Aberdeen, but now I almost always have a couple of them when giving 
a presentation.

I apparently also forgot to lift this part over to the MIRA manual, the 
screenshots alone are first-class eye-openers: for some data sets where the 
people prepped the DNA without waiting for the end of the exponential phase, 
One can see a nice "smiley" shape of the coverage when mapped against a 
reference genome which has the origin of duplication at one of the ends.

For some data sets, the coverage at the origin (and several dozen, if not 
hundred kb to each site) can reach 2.5x to 3x the coverage at the 180° 
position. For de-novo assemblers which try to use kmer statistics, this will 
have an impact. Well, it has at least on MIRA. However, it also has an impact 
for mapping assemblies when one want to find duplicated regions: suddenly the 
whole region around the origin is marked at duplicated. Not good.

> I don't recall coming across this advice before - if you have any references
> that would be educational.

I recall having read a paper where this was mentioned, but I have a hard time 
remembering which one exactly. I think it was one of the earlier papers which 
used 454 & Illumina to do assemblies, must have been 2008 or 2009. We first saw 
this effect internally when we had a sequencing of one of our bugs done with 
454 in 2007, and when we complemented the data with Illumina shortly after it 
was even more visible.

Since then I've seen it on a couple of data sets from 4 or 5 organisms (slow 
and fast growers), and I have a data set from a lower eukaryote where I think I 
have a slight smile shape on some chromosomes, too.

> 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?

Nope, that is all done by the people in the labs and I have almost no knowledge 
how they do it. I just tell them "not in exponential phase!" Oh, and if I 
interpret one of the later data sets right: do not try to pop open the cells 
through mechanical shearing (beads or whatever they used). The DNA coming out 
of one of such an experimental run was incredibly degraded (we got a warning 
from our sequencing provider) an plain awful ... the mapping of that data set 
wasn't anywhere near a more or less smooth coverage like I usually get, it was 
a real roller-coaster.

B.


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