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Methods

Gene discovery and annotation using LCM-454 transcriptome sequencing

¹ Bioinformatics and Computational Biology Graduate Program, Iowa State University, Ames, Iowa 50010, USA; ² Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa 50010, USA; ³ Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA; ⁴ Interdepartmental Plant Physiology Graduate Major and Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50010, USA; ⁵ Department of Agronomy and Center for Plant Genomics, Iowa State University, Ames, Iowa 50010, USA

454 DNA sequencing technology achieves significant throughput relative to traditional approaches. More than 261,000 ESTs were generated by 454 Life Sciences from cDNA isolated using laser capture microdissection (LCM) from the developmentally important shoot apical meristem (SAM) of maize (Zea mays L.). This single sequencing run annotated >25,000 maize genomic sequences and also captured 400 expressed transcripts for which homologous sequences have not yet been identified in other species. Approximately 70% of the ESTs generated in this study had not been captured during a previous EST project conducted using a cDNA library constructed from hand-dissected apex tissue that is highly enriched for SAMs. In addition, at least 30% of the 454-ESTs do not align to any of the 648,000 extant maize ESTs using conservative alignment criteria. These results indicate that the combination of LCM and the deep sequencing possible with 454 technology enriches for SAM transcripts not present in current EST collections. RT-PCR was used to validate the expression of 27 genes whose expression had been detected in the SAM via LCM-454 technology, but that lacked orthologs in GenBank. Significantly, transcripts from 74% (20/27) of these validated SAM-expressed "orphans" were not detected in meristem-rich immature ears. We conclude that the coupling of LCM and 454 sequencing technologies facilitates the discovery of rare, possibly cell-type-specific transcripts.

⁷ Corresponding author.

E-mail schnable{at}iastate.edu; fax (515) 294-5256.

[The sequence data from this study have been submitted to GenBank under accession nos. DW724699–DW985434.]

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5145806

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