Abstract
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.
Highlights
The deep subseafloor biosphere represents a frontier for the discovery of new microbial life, and for investigations of the extent, versatility, and perseverance of life on earth
Experimentation revealed that the number of polymerase chain reaction (PCR) cycles for random amplification metagenomic PCR (RAMP) could be increased to as much as 50, but increasing to 60 cycles resulted in high molecular weight amplification products in the negative controls (Figure 1D)
Further experimentation with the qPCR reaction suggested that the PicoGreen added to the qPCR reaction for quantification of the product may have been interfering in some way with the RAMP reaction
Summary
The deep subseafloor biosphere represents a frontier for the discovery of new microbial life, and for investigations of the extent, versatility, and perseverance of life on earth. There are many challenges in studying this community of microorganisms, and the past 20 years of study have only begun to produce an understanding of this vast and complex ecosystem. Investigations to date suggest that many of these microbes appear to be only distantly related to those we know from the study of surface environments (Sørensen et al, 2004; Inagaki et al, 2006; Lipp et al, 2008). Cultivation studies have produced some useful results (Bale et al, 1997; Mikucki et al, 2003; Toffin et al, 2004), but the majority of microbes in this environment (as well as most microbes on Earth) still evade cultivation efforts. Cultivation-independent methods such as polymerase chain reaction (PCR) amplification and subsequent sequencing directly from environmental DNA hold great promise, and have provided the majority of the information obtained to date (Jørgensen and Boetius, 2007; Orcutt et al, 2011); there are still many challenges to overcome in utilizing these methods to their full potential
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