Next Generation Genomic Sequencing Methods
First Claim
1. A rapid and sensitive method of identifying a nucleic acid sequence motif of an organism comprising:
- providing multiple nucleic acid samples wherein each sample is obtained from a different strain or serotype of the organism;
amplifying sequences of the multiple nucleic acid samples by PCR;
obtaining sequence information of the amplified sequences by next generation sequencing;
determining a polymorphism in the genome of at least one strain or serotype from the sequence information obtained; and
correlating the polymorphism identified with a phenotype or genome location of the at least one strain or serotype to identify the motif.
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Abstract
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.
46 Citations
37 Claims
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1. A rapid and sensitive method of identifying a nucleic acid sequence motif of an organism comprising:
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providing multiple nucleic acid samples wherein each sample is obtained from a different strain or serotype of the organism; amplifying sequences of the multiple nucleic acid samples by PCR; obtaining sequence information of the amplified sequences by next generation sequencing; determining a polymorphism in the genome of at least one strain or serotype from the sequence information obtained; and correlating the polymorphism identified with a phenotype or genome location of the at least one strain or serotype to identify the motif. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for rapidly determining a complete sequence of a target gene or genome comprising:
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producing a series of amplicons by performing a single polymerase chain reaction (PCR) of the target in a heat-stable aqueous mixture containing;
a mix of;
(i) a polymerase, (ii) deoxynucleotide tri phosphates comprising amounts of dATP, dCTP, dGTP and/or dTTP;
(iii) a chelating agent;
(iv) a salt;
(viii) a buffer;
(ix) a stabilizing agent; and
(x) a plurality of primer pairs wherein each primer of the plurality of primer pairs has a similar annealing temperature;sequencing each of the series of amplicons produced by next generation sequencing in a single reaction, and correlating the sequences of the amplicons and constructing the complete sequence of the target gene or genome. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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24. A method for determining the sequence of a nucleic acid target in one cycle of steps comprising:
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providing a sample containing the nucleic acid target; performing a polymerase chain reaction on the nucleic acid of the sample to produce a series of amplicons, wherein the reaction comprises a heat-stable composition comprising; a polymerase;
a mix of deoxynucleotide triphosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP;
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 an annealing temperature within 5°
C.;sequencing each of the series of amplicons produced by NGS, and correlating the sequences of the amplicons and constructing the sequence of the nucleic acid target. - View Dependent Claims (25, 26, 27, 28, 29)
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- 30. A mixture comprising multiple pairs of nucleic acid primers wherein, upon subjecting the mixture to a polymerase chain reaction in association with a nucleic acid target, the nucleic acid primers hybridize to the nucleic acid target at about the same temperature and generates a collection of amplicons, wherein each amplicon of the collection is about 500 to 2,000 nucleotides in length, such that the entire sequence of the target is represented in the resulting collection of amplicons.
Specification