Plant Physiology Preview
Published on March 9, 2007; 10.1104/pp.107.096677
OPEN ACCESS ARTICLE
Received January 26, 2007
Accepted February 27, 2007
Sampling the Arabidopsis Transcriptome with Massively-Parallel Pyrosequencing
Andreas P.M. Weber , Katrin L. Weber , Kevin Carr , Curtis Wilkerson , and John B. Ohlrogge *
Department of Plant Biology, Michigan State University, East Lansing, MI 48824-1312, USA; Bioinformatic Support Core, Research Technologies Support Facility, Michigan State University, East Lansing, MI 48824, USA
* Corresponding author; email: ohlrogge{at}msu.edu.
Massively-parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively-parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts we analyzed mRNA from Arabidopsis seedlings. Two sequencing runs yielded 541,852 ESTs after quality control. Mapping of the ESTs to the Arabidopsis genome and to TAIR7 cDNA models indicated: 1) massively-parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10% but increased the overall sequence coverage by 50%. 2) Mapping of the ESTs to their predicted full length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium and long transcripts were equally represented. 3) 16,698 of the ESTs that mapped to the genome are not represented in the existing dbEST database. In some cases the ESTs provide the first experimental evidence for transcripts derived from predicted genes and for at least 60 locations in the genome pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together the results indicate massively-parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of non-model plants with less complete genomic information.
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