NEW YORK (GenomeWeb News) – Scientists exploring a marine microbial community have developed a way to combine gene expression analysis with metagenomics, according to research published today.
Researchers from the Massachusetts Institute of Technology and Pennsylvania State University used a combination of RNA amplification and next-generation sequencing to look at the gene expression and metagenomics of a marine, microbial community in the North Pacific.
Their work, published in the early, online edition of the Proceedings of the National Academy of Sciences today, confirmed some previous findings, but also revealed some surprises — including widely expressed unknown or hypothetical genes.
“So far in the microbial community, we have looked only at who is there,” lead author Jorge Frias-Lopez, an environmental engineer at the Massachusetts Institute of Technology, told GenomeWeb Daily News. For this study, he said, the researchers wanted to go a step further: trying to understand what the organisms are doing.
Frias-Lopez and his colleagues focused on an entire community in the North Pacific Subtropical Gyre. In order to increase the number of genes they could detect, he explained, they used RNA amplification to increase their overall signal. This involved adding poly-A tails to the bacterial RNA and then doing reverse transcription, an approach that increased the signal by about a thousand-fold.
Using a Roche/454 Life Sciences GS 20 sequencer to capture both cDNA and genomic DNA, they next studied the sequence diversity by comparing all of the sequences to the National Center for Biotechnology Information’s nonredundant protein database. “What we did was normalize against the amount of DNA we had,” Frias-Lopez explained.
To verify their results, they also used RT-qPCR and qPCR. In addition, throughout the study they calibrated and verified their broader microbial community results against Prochlorococcus, a well characterized and highly abundant marine cyanobacteria for which genome and expression data were already available. And because Frias-Lopez and his colleagues had access to Affymetrix custom-designed arrays of Prochlorococcus genes, they were able to assess the amplification method in that organism before applying it to the larger microbial community.
They also tested some of the approaches using samples taken from the Hawaii Ocean time series station in the North Pacific Subtropical Gyre, a region previously subjected to metagenomics, for which databases already exist.
Not surprisingly, their analysis revealed a large number of genes involved in photosynthesis, fixing carbon, and acquiring nitrogen. Interestingly, though, the gene coding for one photo-active pigment, protorhodopsin, was especially abundant.
There were other surprises as well. For instance, at the whole-community level, they saw many new and hypothetical genes with unknown functions that were expressed at high levels. Similarly at a Prochlorococcus level, they saw that many highly expressed genes correlated with parts of the genome representing hypothetical ORFs.
“The fact that a large fraction of cDNA reads were not present in the available databases, including the GOS database, indicates that we have just scratched the surface of the microbial diversity present in the ocean,” the authors wrote.
With increasing interest in community metagenomics, Frias-Lopez said, this new integration of gene expression analysis expands the approach and “opens the possibility of doing these kinds of studies in the field.”
Now that they have verified their technique, he and his colleagues plan to use the gene expression metagenomics approach to make more complete community profiles, for instance, looking at gene expression in marine microbial communities over time.