Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing.

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.

journal_name

Mol Ecol

journal_title

Molecular ecology

authors

Vera JC,Wheat CW,Fescemyer HW,Frilander MJ,Crawford DL,Hanski I,Marden JH

doi

10.1111/j.1365-294X.2008.03666.x

subject

Has Abstract

pub_date

2008-04-01 00:00:00

pages

1636-47

issue

7

eissn

0962-1083

issn

1365-294X

pii

MEC3666

journal_volume

17

pub_type

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