Project II.2.3e: Characterizing communities - Massively parallel Tag Pyrosequencing with bar-coded primers for high-throughput a
Project Name: II.2.3e Characterizing communities - Massively parallel Tag Pyrosequencing with bar-coded primers for high-throughput analysis of microbial community composition
Project Lead: Byron Crump
Project Description
Our goal is to develop a method for high-throughput assessments of microbial community composition using Tag Pyrosequencing of bacterial 16S rRNA genes that are PCR-amplified from hundreds of DNA samples using bar-coded primers. Tag Pyrosequencing uses a new massively parallel DNA sequencing technology to collect sequences from hundreds of thousands of PCR amplicons in a single two-day run. A recent advance in this technique uses uniquely bar-coded primers which can be applied to up to ~1100 samples. PCR products are combined and sequenced in a single pyrosequencing run, and bar codes allow easy sorting of sequences. When applied to 286 samples, Hamady et al. (2008) collected ~1,500 DNA sequences per sample. Compare this to DGGE in which 20 to 50 bands are identified per sample, and acquiring DNA sequences from bands is labor intensive. dThe data produced, even from a single sequencing run, will increase the CMOP database of rRNA gene sequences by one or two orders of magnitude. It will also provide phylum and genus-level diversity information and insight into rare populations undetectable with DGGE.
Fit in program
The persistence of microbial communities on time scales of decades is unknown. However, microbial communities are extremely diverse, fast-growing, and intimately linked to local environmental conditions, and shifts in the composition of these communities represent a highly sensitive and integrative measure of the state of the microbial food web. Thus, microbial communities may serve as first-responders to the biologically important effects of anthropogenic activities and global climate change. This technique will greatly improve our ability to identify patterns in microbial community composition.
Outcomes
This project will establish a new standard method for CMOP nucleic acid sequencing and data analysis.
