Sampling the Arabidopsis Transcriptome with Massively Parallel Pyrosequencing

Abstract

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 (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 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) Over 16,000 of the ESTs that mapped to the genome were 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 nonmodel plants with less complete genomic information.For approximately 30 years, sequencing of DNA by the dideoxy terminator strategy introduced by Sanger (1977) has provided the basis for almost all available information about nucleotide sequences. Pyrosequencing is an alternative technology that detects the pyrophosphate released during DNA polymerase-catalyzed incorporation of nucleotides. The pyrophosphate liberated with each nucleotide addition can generate light in a reaction coupled to ATP sulfurylase and luciferase.

Gene expression profiling by massively parallel sequencing

“…Interestingly, recent studies also assessed the randomness of nebulization and found more reads in the 5Ј end of the transcript (Bainbridge et al 2006;Emrich et al 2007;Weber et al 2007). Although this bias was not very strong, our focus on the 3Ј ends of the transcripts has probably resulted in an even higher homogeneity of the size distribution of the cDNA fragments analyzed.…”

“…While it would be also possible to sequence full-length cDNAs (Bainbridge et al 2006;Emrich et al 2007;Weber et al 2007) rather than 3Ј ends, we consider our approach more cost-effective, as it requires only a single read per transcript. Furthermore, no adjustment for transcript length needs to be made.…”

Facing the Future of Plant-Insect Interaction Research: Le Retour à la “Raison d'Être”

“…Highly parallel sequencing approaches may soon provide an alternative to microarray construction and analysis for studies of gene expression by allowing investigators simply to ''count'' the number of genes expressed and sequenced (e.g. Weber et al, 2007). Ultimately, it may be possible to compare herbivore and plant genomes with a view to reconstructing the coevolutionary history of particularly intimate interactions in the classical gene-for-gene sense.…”

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

“…These microarrays could not cover all genes in the genome, but more recently, massively parallel cDNA sequencing approaches were applied to C. reinhardtii, overcoming the shortcomings of microarrays (González-Ballester et al, 2010). Likewise, we have chosen a cDNA sequencing-based approach using 454 and Illumina technologies in parallel that allow the generation of large numbers of ESTs of varying abundance, which can be counted to obtain a measure of gene expression (Weber et al, 2007).…”