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. 2007 Jan;17(1):69-73.
doi: 10.1101/gr.5145806. Epub 2006 Nov 9.

Gene discovery and annotation using LCM-454 transcriptome sequencing

Scott J Emrich  1 , W Brad Barbazuk, Li Li, Patrick S Schnable
Affiliations

Gene discovery and annotation using LCM-454 transcriptome sequencing

Scott J Emrich et al. Genome Res. 2007 Jan.

Abstract

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 approximately 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 approximately 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 approximately 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.

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Figures

Figure 1.
Figure 1.
Experimental validation of the expression of orphan genes. (A) Test for genomic DNA contamination of cDNA. Primers that flank a 100-bp intron in the maize beta-tubulin6 (tub6) gene were used to amplify genomic DNA (lane 3), SAM cDNA (lane 2), immature ear cDNA (lane 4), and the complex cDNA pool (lane 5). (B) Examples of orphans with validated expression patterns. Primers designed to amplify (lanes 2–4) MAGI_80343, (lanes 5–7) MAGI_60450, (lanes 8–10) MAGI_75030, and (lanes 11–13) MAGI_30050 were used to amplify (lanes 2, 5, 8, 11) SAM cDNA, (lanes 3, 6, 8, 12) immature ear cDNA, and (lanes 4, 7, 9, 13) the pooled cDNA sample. (C) Summary of RT-PCR results for the 27 orphan genes. (+) Indicates that an RT-PCR product of the correct size was detected. Lane 1 of panels A and B contains the One KB Plus size standard (GIBCO BRL). Because primer dimers present in some lanes were cropped in both panels, the smallest size standard band shown is 200 bp.
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