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      Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol

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          Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

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          Most cited references 36

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          QIIME allows analysis of high-throughput community sequencing data.

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            Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.

            mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.
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              BLAST+: architecture and applications

              Background Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. Results We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. Conclusion The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.

                Author and article information

                Role: Academic Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                8 July 2015
                : 10
                : 7
                Department of Animal Ecology, Evolution and Biodiversity, Ruhr University Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany
                University of Guelph, CANADA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: VE FL. Performed the experiments: VE. Analyzed the data: VE. Contributed reagents/materials/analysis tools: FL VE. Wrote the paper: VE FL.


                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

                Figures: 3, Tables: 1, Pages: 16
                This work was supported by a grant of the Kurt Eberhard Bode Foundation to FL.
                Research Article
                Custom metadata
                The Illumina sequencing data are available via the Short Read Archive (accession numbers SRS731403 and SRS733820).



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