In plants the 18 S is far from reaching the species level, you may have to complete the 18S by another, more variable, locus.

Thanks Alex but as said, it is microbial so am (in principle) not interested in plant (or animal) sequences.

Yes of course but the answer will depend of the level of diversity that you want to reach. As far I know eucaryotes can be indentify up to the species level, I am not certain that you can estimate eucaryotic species diversity with 18S sequences.

For Nematodes and Fungi we are trying primers ITS5 and ITS4 for the ITS region.

Check out this paper in Microbial Ecology:

An Evaluation of 18S rDNA Approaches for the Study of Fungal Diversity in Grassland Soils

J. Hunt, L. Boddy, P. F. Randerson and H. J. Rogers

Page 385 of 385-395

Vol. 47, No. 4 (Apr., 2004)

It seems ITS is good for resolution and 18S for larger taxonomic comparisons. You may need seperate primer sets for fungi and protozoa if you are interested in them.

Hi, if fungi is part of your target organisms, try reaching http://taylorlab.berkeley.edu/lab-directory

I am just not aware of any NGS target for diversity in microbial eukaryotes.

If you want to mine data from whole metagenomic sequencing, you could try using the Ray Meta software (http://genomebiology.com/2012/13/12/R122/abstract) and build you database of single cell eukaryotes genomes. The software allows to profile metagenomes for organisms based on sequence databases. We do it with bacteria and it work very well. The only limit of this method is the extend of your sequence database.

As other people have mentioned, it really depends on which organisms you want to characterize and to what level. Rates of diversification vary taxonomically, so the 18S might not give you the resolution for, say, metazoan species or certain green algal lineages. The ITS region is far more variable and might be a good all-around target, although alignments are tricky. One person suggested probing metagenome databases, which may be a great approach if you aren't interested in characterizing a particular community.

Do you have any idea about what kind of organisms you will find, or do you have any predictions that can help guide your gene selection?

i think sequencing of internal transcribed spacer region (ITS) will helps you. and more over the length of that region is very small than 18S or 28S region. Sequence comparison of the ITS region is widely used in taxonomy and molecular phylogeny. The ITS region is now perhaps the most widely sequenced DNA region.

we work a lot with the v9 region of the SSU/18S, but the database is larger with the v4 region... for now that's what we have! ITS might be a candidate for a more variable gene, but there is a high intragenomic diversity and problems might be difficult to handle. Other genes (COI, etc) are unfortunately not well sampled to date...

Thanks everyone, it seems that doing both V4 and V9 might give good, semi-independent results and that the fragment sizes will be do-able using the increased Illumina fragment sizes.

Hi Mark,

We routinely amplify and sequence the fungal ITS1 region of complex communities (all sorts of human microbiome samples).

The primers we use are described here:

http://www.ncbi.nlm.nih.gov/pubmed/20072605

Characterization of the oral fungal microbiome (mycobiome) in healthy individuals.

Ghannoum MA, et al. PLoS Pathog. 2010 Jan 8;6(1):e1000713. doi: 10.1371/journal.ppat.1000713.

We have also designed a microbial specific 18S primer (fwd primer), to be used with a universal reverse primer.

Here is the ref:

http://www.ncbi.nlm.nih.gov/pubmed/22759449

A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples. Dollive S et al. Genome Biol. 2012 Jul 3;13(7):R60.

We have not worked with these amplicons and Illumina yet, but they work well with 454. The sizes should be compatible enough for you to adapt them to an illumina based protocol, if you need to.

Hope it helps,

Chris

John W. Stiller has done a lot of this kind of work, and done a good job of it. A list of his publications are available here:

http://www.experts.scival.com/reachnc/pubDetail.asp?t=pm&id=34249011707&n=John+W+Stiller&u_id=5004080

For rRNA, I believe he has used 16S, not 18S; and for protein sequences I believe he used tubulin genes and the related bacterial protein, ftsZ.