I'm wondering whether anyone has assembled a database of oomycetes and successfully used on the of high-throughput sequencing platforms to characterize the community structure of this group of organisms in soil.
Hi Matthew! Do you mean a taxonomic marker amplicon sequencing specific to Oomycetes, or tracking them out of a whole metagenome seq run?
Salut, Emilie!
I meant a taxonomic marker amplicon sequencing specific to Oomycetes.
As far as I know, there's no specific taxonomic marker for that group. But you can use protistan 18S rRNA universal primers to perform the sequencing and pick up the sequences of interest as an alternative, though it's not very highly efficient.
The most commonly used marker is 3ndf & euk-v4-r (450bp), it's good for ion-torrent &454 but a bit too long for MiSeq, in which case, if the amplicons (R1&R2) cannot fully overlap, it's gonna be very hard for distance-matrix calculating and OTU picking.
Speaking of the database, there‘s a PR 2 database dedicating to the diversity of protistan SSU genes.
Nonetheless，18S rRNA is somewhat too conservative to generate a reliable phylogenetic estimation albeit the protistan taxonomy is largely based on 18S rRNA sequences. It would be great if you can find other makers.
I want to look in plant roots, so am hoping for a marker that will let me avoid plant DNA, which probably makes up the bulk of the sample.
http://www.earthmicrobiome.org/emp-standard-protocols/18s/
use the protocol from above link.
There are some primers you can replace in protocol that can block plant DNA being amplified or you can sequence deep and remove plant seq bioinformatically.
Hi Mathhew,
I have tried High throughput sequencing of Oomycete community in soil using 454 sequencing. I have targeted ITS1 region for PCR. It works perfectly in Plant DNA background. I am presenting poster on this topic in "First global soil biodiversity conference" in Dijon, France next week. If you are interested, I could send you the poster, once it is finished.
I have send you book of abstract and my poster in your email address.
Full paper is online now. See the link below
http://www.sciencedirect.com/science/article/pii/S0167701215000275
Hi. If you are still interested on this topic. In my former lab, we tried using the 28S primers from Arcate etal 2006 Microbial Ecology 51: 36-50 for pyrosequencing. They worked pretty well on recovering oomycete communities, which were otherwise not recovered by pyrosequencing with universal large subunit primers (LROR-LR3). On the end we didn't explore much more on this topic, but it worked well for the samples we tested.
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