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      Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

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          Abstract

          We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.

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          A large genome center's improvements to the Illumina sequencing system.

          Michael Quail, Iwanka Kozarewa, Frances Smith … (2008)
          The Wellcome Trust Sanger Institute is one of the world's largest genome centers, and a substantial amount of our sequencing is performed with 'next-generation' massively parallel sequencing technologies: in June 2008 the quantity of purity-filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64 gigabases. Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.
          Article 0 comments Cited 301 times – based on 0 reviews     Review now
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            A general method for isolation of high molecular weight DNA from eukaryotes.

            N Blin, D W Stafford (1976)
            A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.
            Article 0 comments Cited 197 times – based on 0 reviews     Review now
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              Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

              Kevin McKernan, Heather E. Peckham, Gina Costa … (2009)
              We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.
              Article 0 comments Cited 177 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Genome Biol
                Title: Genome Biology
                Publisher: BioMed Central
                ISSN (Print): 1465-6906
                ISSN (Electronic): 1465-6914
                Publication date (Print): 2010
                Publication date (Electronic): 8 December 2010
                Volume: 11
                Issue: 12
                Page: R119
                Affiliations
                [1 ]Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
                [2 ]The Marine Biological Laboratory, Woods Hole, MA 02543, USA
                [3 ]BGI-Shenzhen, Shenzhen 518000, China
                [4 ]Epicentre Biotechnologies, 726 Post Road, Madison, WI 53713, USA
                Article
                Publisher ID: gb-2010-11-12-r119
                DOI: 10.1186/gb-2010-11-12-r119
                PMC ID: 3046479
                PubMed ID: 21143862
                SO-VID: f94cb215-f224-49c6-ae0f-4356f2b445be
                Copyright © Copyright ©2010 Adey et al.; licensee BioMed Central Ltd.
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 22 September 2010
                Date revision received : 8 November 2010
                Date accepted : 8 December 2010
                Categories
                Subject: Method

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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