Novel degenerate PCR method for whole-genome amplification applied to Peru Margin (ODP Leg 201) subsurfaces samples

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doi: 10.3389/fmicb.2012.00017
Author(s): Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.
Author Affiliation(s): Primary:
Pennsylvania State University, Department of Geosciences, University Park, PA, United States
Other:
University of Delaware, United States
Volume Title: Frontiers in Microbiology
Source: Frontiers in Microbiology, Vol.3(Article 17), p.1-11. Publisher: Frontiers Research Foundation, Lausanne, Switzerland. ISSN: 1664-302X
Note: In English. 25 refs.; illus., incl. 3 tables
Summary: 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.
Year of Publication: 2012
Research Program: ODP Ocean Drilling Program
Key Words: 22 Environmental Geology; Bacteria; Biosphere; Coliform bacteria; Continental margin; Crust; DNA; Depth; East Pacific; Ecology; Equatorial Pacific; Escherichia; Escherichia coli; Genetics; Habitat; Leg 201; Marine sediments; Microorganisms; Nucleic acids; ODP Site 1229; Ocean Drilling Program; Oceanic crust; Pacific Ocean; Peru; Phylogeny; Sediments; South America; South Pacific; Southeast Pacific
Coordinates: S105900 S105900 W0775800 W0775800
Record ID: 2013048186
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