SNP discovery via 454 transcriptome sequencing

doi:10.1111/j.1365-313X.2007.03193.x

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Wiley InterScience

The Plant Journal

Volume 51 Issue 5, Pages 910 - 918

Published Online: 27 Jul 2007

Published in association with the Society for Experimental Biology

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SNP discovery via 454 transcriptome sequencing

W. Brad Barbazuk ^1,† , Scott J. Emrich ^2,3,† , Hsin D. Chen ⁴ , Li Li ^5,6 and Patrick S. Schnable ^2,4,5,6,7,*

¹ Donald Danforth Plant Science Center, St Louis, MO 63132, USA , ² Interdepartmental Bioinformatics and Computational Biology Graduate Program , ³ Department of Electrical and Computer Engineering , ⁴ Department of Agronomy , ⁵ Interdepartmental Plant Physiology Major , ⁶ Department of Genetics, Development, and Cell Biology , and ⁷ Center for Plant Genomics, Iowa State University, Ames, IA 50011, USA

Correspondence to *(fax +1 515 294 5256; e-mail schnable@iastate.edu).

^† These authors contributed equally to this work.

KEYWORDS

SNPs • ESTs • maize • 454 sequencing • markers

ABSTRACT

A massively parallel pyro-sequencing technology commercialized by 454 Life Sciences Corporation was used to sequence the transcriptomes of shoot apical meristems isolated from two inbred lines of maize using laser capture microdissection (LCM). A computational pipeline that uses the POLYBAYES polymorphism detection system was adapted for 454 ESTs and used to detect SNPs (single nucleotide polymorphisms) between the two inbred lines. Putative SNPs were computationally identified using 260 000 and 280 000 454 ESTs from the B73 and Mo17 inbred lines, respectively. Over 36 000 putative SNPs were detected within 9980 unique B73 genomic anchor sequences (MAGIs). Stringent post-processing reduced this number to > 7000 putative SNPs. Over 85% (94/110) of a sample of these putative SNPs were successfully validated by Sanger sequencing. Based on this validation rate, this pilot experiment conservatively identified > 4900 valid SNPs within > 2400 maize genes. These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

Received 20 November 2006; revised 23 April 2007; accepted 11 May 2007.

DIGITAL OBJECT IDENTIFIER (DOI)

10.1111/j.1365-313X.2007.03193.x About DOI