By Ben Butkus
The National Institutes of Health this week awarded grants to a pair of technologies designed to enable whole-genome amplification of single cells for downstream analysis by sequencing as part of the Human Microbiome Project.
The first grant, worth $538,000 over two years and awarded to researchers from GE Global Research, will support the development of a whole-genome DNA amplification method based on multiple displacement amplification that will enable high-throughput DNA sequencing of entire genomes from single cells, eliminating the requirement to purify and culture each isolate, the grant's abstract states.
The second grant, good for $440,000 over two years, will enable Stanford University scientists to develop an approach for plating, selecting, and amplifying whole genomic DNA from individual microbial cells in a hydrogel matrix, according to the grant's abstract.
And although the two grants collectively represent a little under $1 million of the approximately $42 million worth of grants recently awarded by the NIH for human microbiome research, they may help address many of the inherent technical difficulties of isolating, amplifying, and sequencing genetic material from microorganisms that colonize various parts of the human body.
"The biggest problem with human microbiome research is that all of the organisms that are growing on us are growing in communities," John Nelson, a molecular biologist in GE's Biosciences lab and principal investigator on the GE grant, told PCR Insider.
Many of those organisms cannot be cultured, but single cells can be isolated and their genetic material collected. However, a sequencing run on a next-generation sequencer may require as much as 20 micrograms of starting material, but there may only be femtograms available in single bacteria, Nelson said.
"If you sequence it as a mixture, it creates a nightmare for the people who are trying to assemble their contigs and figure out which piece comes from where, especially if you have related organisms in the mixture," Nelson said.
To solve this, Nelson said, "the idea is that if someone can pull out a single bacterium and put it in a tube; if we can pop that bacteria open gently, take the double-stranded DNA … and drop it into our whole-genome amplification kit and make 20 micrograms of DNA, then that alleviates the need to grow up a pure culture of that organism."
The WGA kit to which Nelson referred is GE Healthcare's GenomiPhi, based on the multiple displacement amplification, or MDA, technique co-developed by Nelson and current J. Craig Venter Institute researcher Roger Lasken.
MDA involves annealing random hexamer primers to a template and synthesizing DNA using Φ29 DNA polymerase. The reaction is isothermal and performed at a constant temperature of 30º C.
The MDA method is often used to amplify genomic DNA from small samples and single cells, but over the past few years Nelson and colleagues have been developing a version of the method that is faster and more sensitive.
This has involved modifications such as developing a DNA-free reagent system to address potential foreign DNA contamination; and investigating whether technologies for repairing environmentally damaged DNA for forensic analysis can also be applied to create a better template for the MDA reaction, Nelson said.
"There are a couple of different things we want to try out, and it's just a matter of putting them in a workflow and seeing if we get better sequence," he said.
The GE grant abstract mentions the Broad Institute as a collaborator on the project. Nelson said that the institute's likely role will be to conduct whole-genome sequencing following amplification using the improved GenomiPhi kit.
"If it works, they're certainly going to be one of the first test sites to have the reagent," Nelson said.
A GE spokesperson said that if the revamped method is successful, GE Healthcare's Life Sciences business would then "assess next steps related to commercialization and market opportunities."
The Matrix
Meantime, a group led by Ronald Davis at Stanford University will use its two-year, $440,000 NIH grant to plate, select, and amplify DNA from individual microbial cells using a hydrogel matrix.
Davis was not immediately available for comment. However, according to the NIH grant abstract, the hydrogel matrix approach will allow researchers to spatially isolate and amplify polymerase colonies, also known as polonies, using solid-phase PCR; which in turn "will help expand the reference library of whole genome sequences from the human microbiome."
The grant abstract also states that the group has shown in lab experiments that its WGA method, based on tagged random hexamer PCR, or T-PCR, is sensitive to one genome equivalent, or 1 to 5 femtograms, of DNA.
More specifically, the abstract describes a two-step approach of genome tagging and amplification that the group plans to convert into "two compatible solid-phase PCR reactions using thin layers of porous polyacrylamide hydrogel."
In order to amplify whole genomes from single cells, the group will use standard microbiological plating techniques to spatially isolate E. coli cells onto the surface of the hydrogel and then sandwich the cells between the two reaction layers.
The researchers wrote that their method will be optimized for generating "sequencing-ready" DNA from individual isolated cells, with fragment size controlled by PCR extension time.
"Polonies generated by the T-PCR method will be recovered from the hydrogel and characterized by high-throughput [Roche] 454 pyrosequencing to determine genome sequence coverage and any potential amplification biases," the abstract states.
"For the second phase of research, a complex microbial sample from the oral-salivary microbiome will be evaluated using solid-phase polony amplification," the abstract continues. "The potential for a diversity of different cell types requires an added step of cell lysis/selection, and UV photocatalysis will be explored as a means to weaken the microbial cell wall and improve susceptibility to heat lysis during PCR."
The group will then screen resulting polonies by sequencing the 16S rRNA gene to assess microbial diversity of recovered whole genomes. "If successful, this technology will provide a simple and readily accessible approach for spatially isolating and selecting single cells for whole genome amplification," the researchers wrote.