Award Abstract #0110528
Signature Sequencing for Quantitative Expression Analysis and Gene Discovery

NSF Org:	IOS Division of Integrative Organismal Systems
Initial Amendment Date:	September 24, 2001
Latest Amendment Date:	August 30, 2002
Award Number:	0110528
Award Instrument:	Continuing grant
Program Manager:	Jane Silverthorne IOS Division of Integrative Organismal Systems BIO Directorate for Biological Sciences
Start Date:	October 1, 2001
Expires:	July 31, 2002 (Estimated)
Awarded Amount to Date:	$966641
Investigator(s):	Blake Meyers meyers@dbi.udel.edu (Principal Investigator)
Sponsor:	University of California-Davis OR/Sponsored Programs Davis, CA 95618 530/754-7000
NSF Program(s):	PLANT GENOME RESEARCH PROJECT
Field Application(s):
Program Reference Code(s):	BIOT, 9251, 9183, 9109, 1684, 1329
Program Element Code(s):	1329

ABSTRACT

The primary goal of this project is to demonstrate the utility of a novel technology called 'massively parallel signature sequencing' (MPSS) for the quantification of gene expression in plants. MPSS is a rapid method to produce 17 base pair sequence tags that are precisely representative of the population of messenger RNAs in a given tissue. Approximately eight libraries from diverse plant tissues will be sequenced by MPSS, generating ~500,000 tags per library, for a total of four million tags. The 17-bp tag is derived from the 3' end of a messenger RNA or 'transcript' and provides a virtually unique, experimentally derived identifier for each expressed gene. The number of identical tags in a library for a given gene is precisely indicative of the level of expression of that gene. The MPSS sequence data provide quantitative or 'digital' expression information for the entire 'transcriptome', avoiding problems inherent in microarray analysis such as cross-hybridization, pre-selection of probe sequences and low signal. Statistical methods for the analysis of quantitative expression data have demonstrated that these data are robust. In comparison to the limited sets of data derived by the EST and SAGE techniques, these data will be the first large-scale quantitative expression data for plants in the public domain.

The MPSS sequence data is most informative when the tags are compared to either a completely sequenced genome or to large collections of ESTs. To take full advantage of the MPSS technology, the initial libraries will be generated from the model plant Arabidopsis (ecotype Columbia) and the MPSS tags compared to the complete genomic sequence. This comparison identifies the individual genes from which the tags are derived. These data then will be used to: quantify and experimentally confirm gene expression and mRNA transcripts in diverse wildtype and treated plant tissues (including shoot, root, inflorescence, silique, anthers, callus); estimate the frequency of alternative polyadenylation in plant tissues; study co-regulated gene pairs and estimate promoter strength and tissue specificity; assess global transcriptional changes in the disease resistance response.

Sequence tags generated through this project will be accessible via the web, and the interface will provide tools for data analyses. These data also will be placed in public expression databases (such as those at the National Center for Biotechnology Information, 'NCBI') for comparison to microarray data, facilitating the expression analysis of either any single gene or all genes present in these libraries. These experiments constitute a proof of concept for massively parallel signature sequencing in plants for the global analysis of gene expression.

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