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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.
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“These sequences also produced longer unigenes (mean = 581 bp) than those assembled in previous studies for example, butterfly (197 bp) [60], Eucalyptus grandis (247 bp) [11], coral larval (440 bp) [17], lodegpole pine (500 bp) [10]) and whitefly (clusters: 372 bp; singletons: 265 bp) [27]).”
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Sorted by citations
RNA-Seq: a revolutionary tool for transcriptomics
Wang, Z., Gerstein, M., & Snyder, M. (2009) · Nature Reviews Genetics · doi:10.1038/nrg2484 · Cited by 13,174
Applications of next-generation sequencing technologies in functional genomics
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“Further, unlike the shorter reads generated using the Illumina or SOLiD sequencers, the length of the 454 reads allows for interpretation of sequences generated from species lacking a genome sequence or extensive transcriptome sequences for comparison (e.g., [66]).”
MethodApplications of next generation sequencing in molecular ecology of non-model organisms
Ekblom, R., & Galindo, J. (2010) · Heredity · doi:10.1038/hdy.2010.152 · Cited by 1,118
“…genome assembly Reference Wasp (Polistes metricus) 454 X X X Toth et al., 2007 Glanville fritillary butterfly (Melitaea cinxia) 454 X X X X Vera et al., 2008; Wheat, 2010 Garden pea (Pisum sativum) 454 X Bräutigam et al., 2008 Bigmouth bass (Micropterus salmoides) 454 X Garcia-Reyero et…”
BackgroundNext-generation sequencing technologies and their implications for crop genetics and breeding
Varshney, R. K., Nayak, S. N., May, G. D., & Jackson, S. A. (2009) · Trends in biotechnology · doi:10.1016/j.tibtech.2009.05.006 · Cited by 953
Phylogeography’s past, present, and future: 10 years after Avise, 2000
Hickerson, M. J., Carstens, B. C., Cavender-Bares, J. M., Crandall, K. A., Graham, C. H., Johnson, J. B., Rissler, L. J., Victoriano, P. F., & Yoder, A. D. (2009) · Molecular Phylogenetics and Evolution · doi:10.1016/j.ympev.2009.09.016 · Cited by 672
“As genomic data become available for non-model organisms (Vera et al., 2008), comparative phylogeographic studies will allow identification of different locus-specific divergent selection patterns (Ellegren and Sheldon, 2008; Luikart et al., 2003; MacCallum and Hill, 2006) between pairs of…”
Background
Metadata
- Authors
- VERA, J. C., WHEAT, C. W., FESCEMYER, H. W., FRILANDER, M. J., CRAWFORD, D. L., HANSKI, I., & MARDEN, J. H.
- Affiliations
- Pennsylvania State University (US)
- University of Helsinki (FI)
- University of Miami (US)
- Corresponding author
- VERA, J. CRISTOBAL
- Venue
- Molecular Ecology
- Publisher
- Wiley
- Published
- 2008-03-25
- Volume
- 17
- Issue
- 7
- Pages
- 1636-1647
- ISSN
- 0962-1083 (ISSN-L)
- 1365-294X
- OpenAlex
- W1985484001
- Cited by
- 714
- References
- 0
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 5 · 2023: 2 · 2022: 3 · 2021: 35 · 2020: 24 · 2019: 13 · 2018: 12 · 2017: 16 · 2016: 17 · 2015: 41 · 2014: 75 · 2013: 81 · 2012: 101
- FWCI
- 38.76
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Molecular Ecology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Science Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Agricultural and Biological Sciences › Insect Science › Lepidoptera: Biology and Taxonomy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Chromosomal and Genetic Variations
- Keywords
- Melitaea cinxia, transcriptome assembly, de novo assembly, 454 pyrosequencing, non-model organisms, expressed sequence tags, oligonucleotide microarrays, single nucleotide polymorphisms, functional genomics
- MeSH terms
- Animals, Base Sequence, Butterflies, Sequence Alignment, Alternative Splicing, Sequence Analysis, DNA, DNA, Complementary, Expressed Sequence Tags, Oligonucleotide Array Sequence Analysis, Polymorphism, Single Nucleotide, Gene Expression Profiling
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