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Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing

Authors

  • J. CRISTOBAL VERA,

    1. Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA,
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    • J.C. Vera and C.W. Wheat contributed equally to this work.

  • CHRISTOPHER W. WHEAT,

    1. Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA,
    2. Department of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 1 PL 65, 00014 Helsinki, Finland,
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    • J.C. Vera and C.W. Wheat contributed equally to this work.

  • HOWARD W. FESCEMYER,

    1. Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA,
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  • MIKKO J. FRILANDER,

    1. Institute of Biotechnology, University of Helsinki, Viikinkaari 9 PL 56, 00014 Helsinki, Finland,
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  • DOUGLAS L. CRAWFORD,

    1. Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA
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  • ILKKA HANSKI,

    1. Department of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 1 PL 65, 00014 Helsinki, Finland,
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  • JAMES H. MARDEN

    1. Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA,
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J. Cris Vera, Fax: 814-865-9131; E-mail: jcv128@psu.edu

Abstract

We present a de novo assembly of a eukaryote transcriptome using 454 pyrosequencing data. The Glanville fritillary butterfly (Melitaea cinxia; Lepidoptera: Nymphalidae) is a prominent species in population biology but had no previous genomic data. Sequencing runs using two normalized complementary DNA collections from a genetically diverse pool of larvae, pupae, and adults yielded 608 053 expressed sequence tags (mean length = 110 nucleotides), which assembled into 48 354 contigs (sets of overlapping DNA segments) and 59 943 singletons. blast comparisons confirmed the accuracy of the sequencing and assembly, and indicated the presence of c. 9000 unique genes, along with > 6000 additional microarray-confirmed unannotated contigs. Average depth of coverage was 6.5-fold for the longest 4800 contigs (348–2849 bp in length), sufficient for detecting large numbers of single nucleotide polymorphisms. Oligonucleotide microarray probes designed from the assembled sequences showed highly repeatable hybridization intensity and revealed biological differences among individuals. We conclude that 454 sequencing, when performed to provide sufficient coverage depth, allows de novo transcriptome assembly and a fast, cost-effective, and reliable method for development of functional genomic tools for nonmodel species. This development narrows the gap between approaches based on model organisms with rich genetic resources vs. species that are most tractable for ecological and evolutionary studies.

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