Developing and Applying Methodologies to Characterise Biodiversity Using Ancient and Degraded DNA

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity. Citation: Murray DC, Bunce M, Cannell BL, Oliver R, Houston J, et al. (2011) DNA-Based Faecal Dietary Analysis: A Comparison of qPCR and High Throughput Sequencing Approaches. PLoS ONE 6(10): e25776. doi:10.1371/journal.pone. 0025776 Editor: Carles Lalueza-Fox, Institut de Biologia Evolutiva Universitat Pompeu Fabra, Spain Received July 29, 2011; Accepted September 9, 2011; Published October 6, 2011 Copyright: 2011 Murray et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was obtained from the Foundation for National Parks and Wildlife, the Department of Environment and Conservation, and the Centre for Fish and Fisheries Research at Murdoch University. NEW received support from the Robert Hammond Research Studentship and MB from the Australian Research Council grant FT0991741. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.bunce@murdoch.edu. au