Browsing by Author "Carvalho, Gary"
Now showing 1 - 5 of 5
Results Per Page
Sort Options
- Isolation of marine meiofauna from sandy sediments From decanting to DNA extractionPublication . Fonseca, V. G.; Packer, Margaret; Carvalho, Gary; Power, Deborah; Lambshead, John; Creer, S.This protocol describes the separation of marine meiofauna from sediment and subsequent environmental DNA extraction. In this study meiofauna samples were taken with a 45 mm core from the upper 5 to 10 cm of sediment layer. Separation from sediment was achieved using a decantation process followed by isolation from fine silt using repetitive centrifugation steps with a 1.16 specific gravity (sg) LUDOX-TM solution. Meiofauna were deliberately separated from macrofauna by using a 1 mm sieve on top of a bottle-top sterile 45 !M sieve. High quality DNA was subsequently obtained using the QIAamp DNA Blood Maxi Kit (Qiagen) with minor adjustments to the manufacturer’s protocol. This procedure allowed efficient isolation of meiofaunal representatives from marine sediments and also extraction of high quality environmental DNA that can be used for downstream metagenetic analysis.
- Life in a drop: sampling environmental DNA for marine fishery management and ecosystem monitoringPublication . Gilbey, John; Carvalho, Gary; Castilho, Rita; Coscia, Ilaria; Coulson, Mark W.; Dahle, Geir; Derycke, Sofie; Francisco, Sara M.; Helyar, Sarah J.; Johansen, Torild; Junge, Claudia; Layton, Kara K. S.; Martinsohn, Jann; Matejusova, Iveta; Robalo, Joana I.; Rodriguez-Ezpeleta, Naiara; Silva, Goncalo; Strammer, Ilona; Vasemagi, Anti; Volckaert, Filip A. M.Science-based management of marine fisheries and effective ecosystem monitoring both require the analysis of large amounts of often complex and difficult to collect information. Legislation also increasingly requires the attainment of good environmental status, which again demands collection of data to enable efficient monitoring and management of biodiversity. Such data is traditionally obtained as a result of research surveys through the capture and/or visual identification of organisms. Recent years have seen significant advances in the utilisation of environmental DNA (eDNA) in the marine environment in order to develop alternative cost-effective ways to gather relevant data. Such approaches attempt to identify and/or quantify the species present at a location through the detection of extra-organismal DNA in the environment. These new eDNA based approaches have the potential to revolutionise data collection in the marine environment using non-invasive sampling methods and providing snapshots of biodiversity beyond the capacity of traditional sampling. Here we present a non-technical summary of different approaches in the field of eDNA, and emphasise the broad application of this approach, with value for the governance and management of marine aquatic ecosystems. The review focuses on identifying those tools which are now readily applicable and those which show promise but are currently in development and require further validations. The aim is to provide an understanding of techniques and concepts that can be used by managers without genetic or genomic expertise when consulting with specialists to perform joint evaluations of the utility of the approaches.
- Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analysesPublication . Fonseca, V. G.; Nichols, B.; Lallias, D.; Quince, C.; Carvalho, Gary; Power, Deborah; Creer, S.Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.
- Second-generation environmental sequencing unmasks marine metazoan biodiversityPublication . Fonseca, V. G.; Carvalho, Gary; Sung, W.; Johnson, Harriet F.; Power, Deborah; Neill, Simon P.; Packer, Margaret; Blaxter, M. L.; Lambshead, P. J. D.; Thomas, W. K.; Creer, S.Biodiversity is of crucial importance for ecosystem functioning, sustainability and resilience, but the magnitude and organization of marine diversity at a range of spatial and taxonomic scales are undefined. In this paper, we use second-generation sequencing to unmask putatively diverse marine metazoan biodiversity in a Scottish temperate benthic ecosystem. We show that remarkable differences in diversity occurred at microgeographical scales and refute currently accepted ecological and taxonomic paradigms of meiofaunal identity, rank abundance and concomitant understanding of trophic dynamics. Richness estimates from the current benchmarked Operational Clustering of Taxonomic Units from Parallel UltraSequencing analyses are broadly aligned with those derived from morphological assessments. However, the slope of taxon rarefaction curves for many phyla remains incomplete, suggesting that the true alpha diversity is likely to exceed current perceptions. The approaches provide a rapid, objective and cost-effective taxonomic framework for exploring links between ecosystem structure and function of all hitherto intractable, but ecologically important, communities.
- Ultrasequencing of the meiofaunal biosphere practice, pitfalls and promisesPublication . Creer, S.; Fonseca, V. G.; Porazinska, D. L.; Giblin-Davis, R. M.; Sung, W.; Power, Deborah; Packer, Margaret; Carvalho, Gary; Blaxter, M. L.; Lambshead, P. J. D.; Thomas, W. K.Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.