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      Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

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          Abstract

          High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

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          Most cited references29

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          An integrated semiconductor device enabling non-optical genome sequencing.

          The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
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            Accurate determination of microbial diversity from 454 pyrosequencing data.

            We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.
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              A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes

              Background Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes. Methodology/Principal Findings We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments. Conclusions/Significance Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                7 February 2014
                : 9
                : 2
                : e87624
                Affiliations
                [1 ]Department of Biology, University of Bergen, Bergen, Norway
                [2 ]Uni Computing, Uni Research AS, Bergen, Norway
                [3 ]Department of Ecology and Natural Resources, NEIKER-Tecnalia, Derio, Spain
                [4 ]Department of Informatics, University of Bergen, Bergen, Norway
                [5 ]Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway
                [6 ]Uni Environment, Uni Research AS, Bergen, Norway
                King Abdullah University of Science and Technology, Saudi Arabia
                Author notes

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

                Conceived and designed the experiments: KH KL AL IJ ET CT. Performed the experiments: KH KL. Analyzed the data: KH KL AL IJ ET CT. Contributed reagents/materials/analysis tools: KH KL AL IJ ET CT. Wrote the paper: KH KL AL IJ ET CT.

                Article
                PONE-D-13-27643
                10.1371/journal.pone.0087624
                3917833
                24516555
                a0e034d3-376c-40f0-bac8-c1a6e9dfe6f3
                Copyright @ 2014

                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.

                History
                : 4 July 2013
                : 18 November 2013
                Page count
                Pages: 10
                Funding
                This work was supported by the Norwegian Research Council, Havet og Kysten, Project 190265/S40. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Computational Biology
                Sequence Analysis
                Ecology
                Community Ecology
                Community Structure
                Ecological Environments
                Marine Environments
                Biodiversity
                Microbial Ecology
                Marine Biology
                Marine Monitoring
                Earth Sciences
                Marine and Aquatic Sciences
                Marine Monitoring

                Uncategorized
                Uncategorized

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