Novel degenerate PCR method for whole-genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples
- 1 Department of Geosciences, Penn State Astrobiology Research Center, Pennsylvania State University, University Park, PA, USA
- 2 School of Marine Science and Policy, College of Earth Ocean and Environment, University of Delaware, Lewes, DE, USA
- 3 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA
A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples.
Keywords: whole-genome amplification, metagenomics, deep biosphere, low biomass, bacteria, archaea, next-gen sequencing
Citation: Martino AJ, Rhodes ME, Biddle JF, Brandt LD, Tomsho LP and House CH (2012) Novel degenerate PCR method for whole-genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples. Front. Microbio. 3:17. doi: 10.3389/fmicb.2012.00017
Received: 14 October 2011; Paper pending published: 03 November 2011;
Accepted: 09 January 2012; Published online: 23 January 2012.
, University of North Carolina at Chapel Hill, USA
Copyright: © 2012 Martino, Rhodes, Biddle, Brandt, Tomsho and House. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.
*Correspondence: Christopher H. House, Department of Geosciences, Penn State Astrobiology Research Center, Pennsylvania State University, 220 Deike Building, University Park, PA 16802, USA. e-mail: firstname.lastname@example.org