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9:47 am
August 19, 2010
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John Dennehy
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I was wondering if we could pcr amplify a region of the mycophage genome
that might serve as an indicator of "phage type" much like 16S rRNA
universal primers does for bacteria? I was thinking of incorporating pcr
into our phage selection process for deciding what phage to sequence.
What do you think? Anyone else considering this?
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9:51 am
August 19, 2010
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Dan Russell
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Post edited 1:53 pm – August 19, 2010 by Dan Russell
It's a good question. So far what we at Pitt (sort of) have is cluster-specific primers.
The idea is you try all pairs (something like 15 sets, one for each
cluster/subcluster) on a new phage's lysate, and you do or don't get a product for
each cluster. If everything works perfectly, you get 14 failed reactions and 1
bright shining positive. In my experience, however, it's more likely that you get
multiple products (at least across different subclusters, and even sometimes across
different clusters) so it's not a definitive answer.
I'm not sure if all the phages have any regions that are similar enough so that one
set of primers could suffice. And you could build some wobble into your primers,
but if you want to directly sequence the product it's got to be a pretty clean
product.
IDing phages reliably and early is a tough issue. Other ideas?
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11:13 am
August 23, 2010
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Dan Russell
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And here's the spreadsheet of the phage/cluster primers we've designed at Pitt (thanks to Victoria Hohenstein!).
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4:30 pm
September 14, 2010
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Dan Russell
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| posts 44 | |
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John got some interesting and useful results from trying to use these primers. It would be interesting to know if anyone else has results to share.
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4:37 pm
February 16, 2011
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Welkin
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How can I use my cluster-specific primers? Why aren't PCR primers great for assigning sub-cluster?
Right now there are several SEA schools that are designing and using cluster
specific primers with varying results. Some of their work has been posted on
our cluster phorum at phagesdb.org/wp/. Schools have been sharing and
swapping phages of different clusters amongst themselves, as well, and we
would like to encourage you to do the same. If you do find that you would
like a particular phage that is only in the Pitt collection, please let us
know and we would be happy to help.
As far as the utility of cluster primers goes, we think that PCR primers can
be very useful in determining an initial assignment for a phage cluster
prior to sequencing (especially if the particle measurements by electron
microscopy bear out the cluster assignment), or to screen a specific lysate
for contaminating phage particles. It may also be possible to test
environmental samples for the presence of phages of specific cluster, which
could lead to some interesting ecology questions: "Do cluster C phages only
come from dry soil?" etc. Of course this would also mean testing the primers
at least in silico on all other known phage sequences to see if you might
accidentally amplify something from, say, a Streptomyces phage.
One caveat to the use of primers for phage clustering is that PCR products
are somewhat limited in their ability to discern subcluster, simply because
subcluster assignments do not remain static as we get new full genome
sequences. For example, last year prior to the sequencing efforts by the SEA
and Pitt, we had only two subclusters in the A cluster; we are now up to
six, with at least three previously sequenced phages being reassigned to one
of the new subclusters. We also gained an I subcluster, two K subclusters,
an L subcluster, and clusters M and N.
All of these were determined using our four metrics for subcluster
assignment (as laid out in Hatfull et al 2010): Average nucleotide identity,
dot plot analysis, gene content, and genome map analysis. None of these can
really be tested solely by PCR product. For this reason, we aren't entering
subcluster assignments into phagesdb.org.
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