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      A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

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          Abstract

          Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

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          AFLP: a new technique for DNA fingerprinting.

          A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
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            Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.

            We constructed error-correcting DNA barcodes that allow one run of a massively parallel pyrosequencer to process up to 1,544 samples simultaneously. Using these barcodes we processed bacterial 16S rRNA gene sequences representing microbial communities in 286 environmental samples, corrected 92% of sample assignment errors, and thus characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.
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              Target-enrichment strategies for next-generation sequencing.

              We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                4 May 2011
                : 6
                : 5
                : e19379
                Affiliations
                [1 ]Institute for Genomic Diversity, Cornell University, Ithaca, New York, United States of America
                [2 ]Computational Biology Service Unit, Cornell University, Ithaca, New York, United States of America
                [3 ]Hard Winter Wheat Genetics Research Unit, United States Department of Agriculture/Agricultural Research Service, Manhattan, Kansas, United States of America
                [4 ]Plant, Soil and Nutrition Research Unit, United States Department of Agriculture/Agricultural Research Service, Ithaca, New York, United States of America
                Temasek Life Sciences Laboratory, Singapore
                Author notes

                Conceived and designed the experiments: RJE JCG QS JAP KK ESB SEM. Performed the experiments: RJE JCG QS JAP KK. Analyzed the data: RJE JCG QS JAP ESB. Contributed reagents/materials/analysis tools: RJE JCG QS JAP ESB SEM. Wrote the paper: RJE JCG QS JAP ESB SEM.

                Article
                PONE-D-10-04702
                10.1371/journal.pone.0019379
                3087801
                21573248
                7e88964d-677e-43c3-994a-8ec49c8df08d
                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
                History
                : 12 November 2010
                : 4 April 2011
                Page count
                Pages: 10
                Categories
                Research Article
                Biology
                Genetics
                Heredity
                Genotypes
                Plant Genetics
                Crop Genetics
                Population Genetics
                Genetic Polymorphism
                Genomics
                Genome Complexity
                Genome Sequencing
                Plant Science
                Agronomy
                Plant Breeding
                Plant Genetics
                Plant Genomics

                Uncategorized
                Uncategorized

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