Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencing

Abstract

Describing the "ORFeome" of an organism, including all major isoforms, is essential for a systems understanding of any species; however, conventional cloning and sequencing approaches are prohibitively costly and labor-intensive. We describe a potentially genome-wide methodology for efficiently capturing novel coding isoforms using RT-PCR recombinational cloning, "deep well" pooling, and a "next generation" sequencing platform. This ORFeome discovery pipeline will be applicable to any eukaryotic species with a sequenced genome.Experimental definition of the complete set of protein-coding transcript sequences ("ORFeome") is fundamental for complete understanding of any organism, but this has not been achieved to date for any metazoan. Adding to the uncertainty, many eukaryotic genes exhibit alternative splicing, leading to a diversity of ORFs encoded by a single gene. Currently, 74% of human multi-exon genes and ~13% of Caenorhabditis elegans genes are predicted to undergo alternative splicing 1,2 . Expansion of the "isoform-space" in more complex organisms may partly explain the paradoxical lack of correlation between organismal complexity and gene number, and underscores the need to efficiently and comprehensively capture the full ORFeome. Historically, determination of intron-exon boundaries in eukaryotes has been addressed mainly by large-scale sequencing of random cDNAs (expressed sequence tags or ESTs) followed by alignment to a reference genomic DNA sequence. Although EST collections are extremely helpful, the human isoform-space remains under-explored. A targeted cloning and full length sequencing strategy could provide the desired information, but is impractically resource-intensive.Correspondence should be addressed to M.V. (marc_vidal@dfci.harvard.edu), K.S.A (kourosh_salehi-ashtiani@dfci.harvard.edu), or F.P.R. (fritz_roth@hms.harvard.edu). 5 These authors contributed equally to this work. Competing interest statementThe authors declare that they have no competing financial interests. Next-generation parallel sequencing technologies, such as the Roche 454 FLX, offer the prospect of sequencing at a much faster pace and lower cost than conventional Sanger-capillary platforms 3 . Most applications described so far have entailed resequencing of megabase-scale genomic DNA fragments 4-7 or of small sequence tags [8][9][10][11] . A disadvantage of the latter approach is that cis-connectivity is lost between the reads; therefore, although the reads can be assembled into contigs, mRNAs can not be assembled unambiguously when splice variants are involved. Sequencing of kilobase-scale DNA fragments from complex pools in which fragments have heterogeneous abundance has not yet been tested, nor has correct assembly of hundreds to thousands of full-length cDNAs in parallel from a complex mixture been proven feasible. NIH Public AccessPrevious and ongoing full-length cDNA isolation projects aim to discover one isoform per gene, without attempting to investigate the depth of "isoform space". Here we describe ...

Transcriptome Analysis of Crucian Carp (Carassius auratus), an Important Aquaculture and Hypoxia-Tolerant Species

“…Full-length cDNA sequences have complete sequences of transcripts including coding regions (CDSs) or all exons and untranslated regions (UTRs), and thus facilitate subsequent studies on genomic structure and functional analysis [25].It is a time consuming and laborious process using the traditional Sanger sequencing method to obtain large collections of full-length cDNAs. Fortunately, the NGS method used in this study and other studies has shown to be an efficient approach to obtain a large number of full-length cDNAs [26]–[27]. In addition, over 7 000 unigenes with 5′ or 3′ UTR are also useful for obtaining full-length cDNA sequences via the primer walking method.…”

Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing

“…First, full-length ORFs corresponding to known and novel isoforms are amplified by reverse transcription followed by PCR (RT-PCR) using gene-specific primers. Pools of resulting RT-PCR products are Gateway-cloned (Walhout et al, 2000) and individual ORFs are sequenced using an NGS-based deep-well approach (Salehi-Ashtiani et al, 2008). Second, Gateway-cloned full-length isoform ORFs are transferred into various expression vectors to allow systematic functional analyses such as binary protein-protein and protein-DNA interaction assays or measurement of enzymatic activities.…”

Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing

“…Recent studies (3)(4)(5)(6)(7) have shown that, by sequencing the mRNA content of cells, one can quantify the expression levels of known genes (by counting how often sequences from a given gene are observed) and refine their boundaries. For example, Nagalakshmi et al (3) studied the Saccharomyces cerevisiae transcriptome by mapping reads to the location of known genes to quantify expression, and to known splice sites to measure their occurrence.…”