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. However, 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. Marine subsurface microorganisms are isolated from the direct energy of sunlight, receive limited nutrients, and sometimes experience extreme pressures and challenging temperatures. 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
); however, there are still many challenges to overcome in utilizing these methods to their full potential.
Among the available cultivation-independent methods, automated metagenomic sequencing via platforms such as Roche 454, is one of the most promising tools for probing the depths of diversity and exploring metabolic capabilities of subsurface microbes. However, even with recent advances in technology, this type of high-throughput sequencing requires, ideally, at least 500
ng of sample DNA (as per Roche 454 protocol). The relatively low concentrations of cells in the marine subsurface coupled with the difficulties of extracting DNA from marine sediment (Webster et al., 2003
) results in quantities of extracted DNA which are often too low for direct, unamplified, metagenomic sequencing.
Over the past couple of decades, researchers have been experimenting with different methods of amplifying genomic DNA. Some of the resulting whole-genome amplification (WGA) methods are modifications to the standard PCR, which reduce its specificity, allowing for a general amplification of DNA. These methods include interspersed repetitive sequence PCR (IRS-PCR; Nelson et al., 1989
), primer-extension-preamplification PCR (PEP-PCR; Zhang et al., 1992
), improved primer-extension-preamplification PCR (I-PEP-PCR; Dietmaier et al., 1999
), degenerate oligonucleotide-primed PCR (DOP-PCR; Telenius et al., 1992
), and long products from low DNA quantities DOP-PCR (LL-DOP-PCR; Kittler et al., 2002
). In addition to the PCR-based methods, a non-PCR method called multiple displacement amplification (MDA) was developed in attempt to overcome problems with the PCR methods, which included incomplete coverage, amplification artifacts, and DNA too short for some applications (Dean et al., 2002
). MDA is an isothermal, strand-displacing reaction employing the phi29 DNA polymerase and random hexamer primers. Several commercial versions of MDA now exist, including the REPLI-g Whole-Genome Amplification Kit (Qiagen) and the GenomiPhi DNA Amplification Kit (GE Healthcare). Two commercial MDA kits, along with PEP-PCR and DOP-PCR, were analyzed for genome coverage bias in a 454 metagenomic sequencing study and all were found to induce significant bias (Pinard et al., 2006
). For both microbial genomes utilized in that study, the MDA reactions resulted in the least bias, followed by PEP-PCR, and lastly, DOP-PCR. These results were consistent with those obtained through a TaqMan
quantitative PCR analysis of eight genes after amplification of human genomic DNA using MDA, DOP-PCR, and PEP-PCR (Dean et al., 2002
As a result, MDA-based techniques are most often the method of choice in applications where bias and coverage are significant concerns. For metagenomic sequencing of subseafloor environmental DNA samples, however, the use of MDA as an amplification technique is often problematic. In particular, the tendency of MDA to synthesize a DNA product even in the absence of added cells, means that a reliable negative control for the amplification reaction is very difficult to achieve (Raghunathan et al., 2005
). These products that form in the negative controls can be as large as 15
kb and greater (Biddle, 2006
). While they have not been sequenced, failure of attempts to PCR amplify specific genes from the products suggests that they are more likely primer-dimer type formations rather than microbial contamination (Biddle, 2006
). This may be a consequence of the competitive nature of the reaction, whereby in the absence of anything else of which to anneal, the random hexamers anneal to each other. If this were the case, the problem would be of greatest significance when sample DNA template is very low, and unable to compete with the hexamers for annealing and amplification. It has been shown that the DNA product formed in negative controls can be avoided if the reaction is monitored closely with a qPCR protocol and stopped before amplification in the negative control begins (Biddle et al., 2011
). However, in many cases, this occurs after less than 2
h reaction time, and stopping the reaction at this length of time may hinder the amplification of sample DNA as well, particularly when the sample had very small amounts of DNA to begin with (Biddle et al., 2011
), as is the case with most deep subseafloor samples.
Due to the difficulties with using available WGA methods for amplifying subseafloor DNA samples, we undertook efforts to develop an alternative method of WGA that may be of use when sequencing low-biomass environmental samples. We hypothesized that the production of DNA in reaction negatives of MDA was a result of the high level of degeneracy of the primers. We recognized, however, that this high level of degeneracy was critical in obtaining amplified DNA with the least amount of bias, which appears to be the reason that the PEP-PCR and MDA methods produce less bias than the PCR methods with more specific primers. Consequently, our strategy was to design a PCR method with primers that would be more degenerate than IRS-PCR and DOP-PCR – and thus applicable to environmental DNA samples, but less degenerate than PEP-PCR and MDA. In addition, with the rapidly increasing use of next generation metagenomic sequencing technology, we aimed to have our new method aid in streamlining the process of preparing samples for metagenomic sequencing using these new technologies. The result was a PCR-based method of WGA utilizing 454 amplicon primers with an attached degenerate region at the 3′ end. We refer to this method as random amplification metagenomic PCR (RAMP). While optimized here for use with Roche 454 sequencing, the developed method may be viewed as a general framework for using PCR for WGA of low-biomass environmental DNA samples in preparation for metagenomic sequencing using next generation sequencing technologies.