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Construction of cDNA. We are testing new methods to rapidly determine the sequence of any dsRNAs that are found in plant samples. The RNA is converted to cDNA by reverse transcription (RT), using a RandomHexPrimer. The first strand cDNA is then purified, the RNA is digested with RNase A, and the the primers are removed. This leaves antiparallel strands of cDNA. These are allowed to anneal and fill-in the ends. A PCR reaction amplifies the annealed strands using the primer sites that were added in the RT reaction. The PCR primers also contain a 4 nt tag that will serve as a key to link the sequence with the original plant sample. Sequence analysis. We will use the new 454 Life Technologies sequencing. Analysis is completed on a microfabricated slide (Margulies et al. Nature 437:378). Individual DNA molecules are captured on beads in an oil droplet emulsion, and an adaptor sequence containing primer binding sites for amplification and sequencing, as well as a key sequence of 4 nt, is added. All of the further reactions, (amplification and sequencing) are carried out on the beads. Twenty samples, each one amplified with its unique primer, will be used on each of 16 strips of a 454 slide. These strips yield about 10,000 sequences of 100-150 nt each (Brendon Hill, 454 Life Sciences, personal communication), so we should have an average of 500 sequences for each sample. This will give us about 7 X coverage (50 ,000 nt) for each sample, if the viral genomes are of expected size (5-10 kb). Bioinformatics. The resulting sequence data will be sorted by slide number, strip number, and tag. This will be the "address" of the sample, matching it to a plant sample. Sequences from each sample will be compiled into contigs, and contigs will be searched against GenBank. Sequence data will be submitted to a unique Taxonomy Node in GenBank annotated as "dsRNA, probably plant virus origin". Sequences will also be maintained in a searchable database to allow for specific queries to answer ecological questions. |
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