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Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

1 , 1 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 8 , 8 , 9 , 10 , 10 , 10 , 9 , 11 , 12 , 11 , 1 , 1 , 13 , 14 , 15 , 15 , 16 , 3 , 17 , 17 , 16 , 16 , 16 , 3 , 3 , 16 , 17 , 2 , 18 , 3 , 1 , 10 , 15 , 12 , 19 , 5 , 20 , 7 , 15 , 21 , 15 , 22 , 14 , 8 , 11 , 23 , 17 , 24 , 2 , 25 , 26 , 6 , 27 , 1 , 1 , 28 , a , 1 , 29

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DNA sequencing, Plant genetics, Genome assembly algorithms

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      Barley ( Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. ‘Morex’ was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

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      The Sequence Alignment/Map format and SAMtools

      Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: Contact:
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        Fast and accurate short read alignment with Burrows–Wheeler transform

        Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: Contact:
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          BEDTools: a flexible suite of utilities for comparing genomic features

          Motivation: Testing for correlations between different sets of genomic features is a fundamental task in genomics research. However, searching for overlaps between features with existing web-based methods is complicated by the massive datasets that are routinely produced with current sequencing technologies. Fast and flexible tools are therefore required to ask complex questions of these data in an efficient manner. Results: This article introduces a new software suite for the comparison, manipulation and annotation of genomic features in Browser Extensible Data (BED) and General Feature Format (GFF) format. BEDTools also supports the comparison of sequence alignments in BAM format to both BED and GFF features. The tools are extremely efficient and allow the user to compare large datasets (e.g. next-generation sequencing data) with both public and custom genome annotation tracks. BEDTools can be combined with one another as well as with standard UNIX commands, thus facilitating routine genomics tasks as well as pipelines that can quickly answer intricate questions of large genomic datasets. Availability and implementation: BEDTools was written in C++. Source code and a comprehensive user manual are freely available at Contact:; Supplementary information: Supplementary data are available at Bioinformatics online.

            Author and article information

            [1 ]Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben , 06466 Seeland, Germany
            [2 ]School of Veterinary and Life Sciences, Murdoch University , Murdoch, Western Australia 6150, Australia
            [3 ]Centre for Comparative Genomics, Murdoch University , Murdoch, Western Australia 6150, Australia
            [4 ]Australian Export Grains Innovation Centre , South Perth, Western Australia 6151, Australia
            [5 ]Department of Agronomy and Plant Genetics, University of Minnesota , St Paul, Minnesota 55108, USA
            [6 ]The James Hutton Institute , Dundee DD2 5DA, UK
            [7 ]European Molecular Biology Laboratory—The European Bioinformatics Institute , Hinxton CB10 1SD, UK
            [8 ]Leibniz Institute on Aging—Fritz Lipmann Institute (FLI) , 07745 Jena, Germany
            [9 ]BioNano Genomics Inc. , San Diego, California 92121, USA
            [10 ]Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research , 78371 Olomouc, Czech Republic
            [11 ]Department of Botany & Plant Sciences, University of California, Riverside , Riverside, California 92521, USA
            [12 ]Department of Computer Science and Engineering, University of California, Riverside , Riverside, California 92521, USA
            [13 ]Department of Agricultural and Environmental Sciences, University of Udine , 33100 Udine, Italy
            [14 ]Green Technology, Natural Resources Institute (Luke), Viikki Plant Science Centre, and Institute of Biotechnology, University of Helsinki , 00014 Helsinki, Finland
            [15 ]Earlham Institute , Norwich NR4 7UH, UK
            [16 ]BGI-Shenzhen , Shenzhen 518083, China
            [17 ]College of Agriculture and Biotechnology, Zhejiang University , Hangzhou 310058, China
            [18 ]Kansas State University, Wheat Genetics Resource Center, Department of Plant Pathology and Department of Agronomy , Manhattan, Kansas 66506, USA
            [19 ]School of Agriculture, University of Adelaide , Urrbrae, South Australia 5064, Australia
            [20 ]Department of Plant and Microbial Biology, University of Minnesota , St Paul, Minnesota 55108, USA
            [21 ]School of Environmental Sciences, University of East Anglia , Norwich NR4 7UH, UK
            [22 ]National Institute of Agricultural Botany , Cambridge CB3 0LE, UK
            [23 ]Department of Biology, Lund University , 22362 Lund, Sweden
            [24 ]Carlsberg Research Laboratory , 1799 Copenhagen, Denmark
            [25 ]Department of Agriculture and Food, Government of Western Australia , South Perth, Western Australia 6150, Australia
            [26 ]Hubei Collaborative Innovation Centre for Grain Industry, Yangtze University , Jingzhou, Hubei 434025, China
            [27 ]School of Life Sciences, University of Dundee , Dundee DD2 5DA, UK
            [28 ]School of Plant Biology, University of Western Australia , Crawley 6009, Australia
            [29 ]German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig , 04103 Leipzig, Germany
            Author notes

            These authors contributed equally to this work


            BAC sequencing and assembly (1H, 3H, 4H): S.B., A.Himmelbach, S.T., M.F., M.G., M.M., U.S. (co-leader), M.P. (co-leader), N.S. (leader); BAC sequencing and assembly (2H, unassigned): D.S., D.H., S.A. (co-leader), M.D.C. (co-leader), M.C. (co-leader), R.W. (leader); BAC sequencing and assembly (5H, 7H): X.Z., R.A.B., Q.Z., C.T., J.K.M., B.C., G.Zhou, F.D., Y.H., S.Y., S.Cao, S.Wang, X.L., M.I.B., P.L., G.Zhang (co-leader), C.Li (leader); BAC sequencing and assembly (6H): S.B., S.Wang, C.Lin, H.L., U.S., M.H. (co-leader), I.B. (leader); BAC sequencing (gene-bearing): M.M.-A., R.O., S.Wanamaker, S.L. (co-leader), T.J.C. (leader); Optical mapping: A.Hastie, H.Š., J.T., H.S., J.V., S.Chan, M.M., N.S., J.D., A.H.S. (leader); Chromosome conformation capture: A.Himmelbach, S.G., M.M. (co-leader), N.S. (leader); Pseudomolecule construction: M.M. (leader), S.B., C.C., D.B., T.S., P.K., N.S., U.S. (co-leader); Validation: L.L., M.B., L.A.-S., A.Houben, J.A.P., N.S., G.J.M., M.M. (leader). All authors read and commented on the manuscript.

            Sci Data
            Sci Data
            Scientific Data
            Nature Publishing Group
            27 April 2017
            : 4
            28448065 5407242 sdata201744 10.1038/sdata.2017.44
            Copyright © 2017, The Author(s)

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