Ion torrent genomic sequencing

摘要

Disclosed is an enhanced method for rapid and cost-effective analysis of sequences of a microorganism by semi-conductor sequencing, preferably ion-torrent sequencing. This method provides for full length analysis and of multiple areas (e.g. genes) of multiple genomes. These methods identify genetic mutations of a particular gene that are responsible for conferring resistance or sensitivity to an antibiotic or other chemical compound. Multiple different species, strains and/or serotypes of a particular organism are rapidly and efficiently screened and mutations identified along with the complete genome of an organism. By selecting primers pairs of similar size and GC content that produce amplicons with sequences spanning the entire genome, a single PCR reaction analyzed by ion torrent methodology can determine the sequence of a complete genome. Methods are useful to sequences the genomes of viral agents, such as influenza virus, and bacterial agents, such as tuberculosis bacteria.

主权项

1. A method for determining a complete sequence of a target gene or genome of a microorganism, wherein the target gene or genome is greater than 1 Mb in length, comprising:
producing a series of amplicons by performing a single polymerase chain reaction (PCR) of the target in an aqueous mixture containing a heat-stable polymerase; a mix of deoxynucleotide triphosphates; a chelating agent; a salt; a buffer; a stabilizing agent; and a plurality of primer pairs wherein each primer of the plurality of primer pairs has a similar annealing temperature and wherein each primer pair is designed to PCR amplify an amplicon representing a portion of the sequence of the nucleic acid target, and the series of amplicons that are PCR amplified represent overlapping portions of the complete sequence of the target; sequencing each of the series of amplicons by semi-conductor sequencing, and correlating the sequences of the amplicons and constructing the complete sequence of the target.