My name is Jeff Norman and I am a doctoral candidate in Jeb Barrett's lab at Virginia Tech.
I've spent most of my Ph.D. studying ammonia-oxidizing microbes, mostly using qPCR to estimate the growth and abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in temperate soil environments. For one chapter of my Ph.D. I hope to estimate the diversity of soil AOA and AOB as well. I had planned on doing this using amplicon-based 454 sequencing of the amoA gene relevant to each group. Though I've had good luck with funding for the rest of my Ph.D., I've had a hard time securing funding for this analysis. I have been looking into other ways of estimating AOA and AOB diversity, and our lab manager alerted me about an interesting procedure called amplicot (the name is based on Cot-curve style analysis of PCR amplicons). Here are two references from Nature Methods about amplicot: 1) Arstila, T.P. 2006. Estimating diversity, the easy way. Nature Methods 3(11): 879-890. 2) Baum, P.D., and J.M. McCune. 2006. Direct measurement of T-cell receptor diversity with Amplicot. Nature Methods 3(11): 895-901. There is also an amplicot website: http://amplicot.ucsf.edu/ The basic principal is pretty simple: First a PCR amplicon is created and combined with SYBR green dye. Then, the product is denatured at high temperature and allowed to re-anneal in a quantitative thermocycler. The annealing time is tracked by fluorescence and used as a metric of sequence diversity (samples with longer annealing times should be more diverse than samples with shorter annealing times). I am interested in using this procedure to estimate relative differences in diversity of AOA and AOB between my samples. Amplicot appeals to me because I have the majority of the necessary materials on hand, and it would therefore be inexpensive for me to perform. Furthermore, I have few enough samples that I could probably squeeze them into one run, and not have to correct for run to run differences, which would potentially make this procedure more complex. Before I go down this road, however, I have a couple of questions for you guys: 1) I have noticed that all the papers citing the original amplicot protocol (Baum and McCune 2006, above) seem to be in immunological journals, and not in ecological journals. Has anyone tried this procedure for environmental samples in the way I'm proposing here? 2) Do you think that a paper based on amplicot analysis would be publishable in ecology/soil journals? I would appreciate answers to these questions along with any additional insight you can provide! Jeff ------------------------------------------------------- Jeff Norman PhD Candidate Department of Biological Sciences Virginia Tech 2119 Derring Hall, Blacksburg VA 24060
