An embodiment of a method for generating a population of amplified concatamer products is described that comprises amplifying a template nucleic acid molecule using a first nucleic acid primer immobilized on a bead substrate and a second nucleic acid primer in solution to generate a population of substantially identical copies of the template nucleic acid molecule immobilized on the bead substrate; and amplifying the population of substantially identical copies of the template nucleic acid molecule using a concatamer primer that comprises a first region complementary to an end region of the population of substantially identical copies of the template nucleic acid molecule and a second region to generate a population of immobilized concatamer products of the substantially identical copies of the template nucleic acid molecule.
An embodiment of a device for automatically executing a process of generating an emulsion containing nucleic acids, amplifying the nucleic acids in the emulsion, breaking the emulsion, and separating and purifying said amplified nucleic acids, is described that comprises an emulsion generation unit for sealing beads to which nucleic acids are bound in a water-in-oil type emulsion; a nucleic acid amplification unit provided with a reaction vessel for amplifying said nucleic acids and a heating and cooling part for heating and cooling the reaction vessel; an emulsion breaking unit for breaking the emulsion after nucleic acid amplification; and a nucleic acid purification unit for recovering said amplified nucleic acids from said emulsion breaking unit.
An embodiment of a method for generating a flow order that minimizes the accumulation of phasic synchrony error in sequence data is described that comprises the steps of: (a) generating a plurality of sequential orderings of nucleotides species comprising a k-base length, wherein the sequential orderings define a sequence of introduction of nucleotide species into a sequencing by synthesis reaction environment; (b) simulating acquisition of sequence data from one or more reference genomes using the sequential orderings, wherein the sequence data comprises an accumulation of phasic synchrony error; and (c) selecting one or more of the sequential orderings using a read length parameter and an extension rate parameter.
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
G06F 19/12 - for modelling or simulation in systems biology, e.g. probabilistic or dynamic models, gene-regulatory networks, protein interaction networks or metabolic networks
G06F 19/20 - for hybridisation or gene expression, e.g. microarrays, sequencing by hybridisation, normalisation, profiling, noise correction models, expression ratio estimation, probe design or probe optimisation
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
G06F 19/24 - for machine learning, data mining or biostatistics, e.g. pattern finding, knowledge discovery, rule extraction, correlation, clustering or classification
4.
System and method for correcting primer extension errors in nucleic acid sequence data
An embodiment of method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises (a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; (b) generating a value for the signal; and (c) correcting the value for the phasic synchrony error using a first parameter and a second carry forward parameter.
G06F 19/20 - for hybridisation or gene expression, e.g. microarrays, sequencing by hybridisation, normalisation, profiling, noise correction models, expression ratio estimation, probe design or probe optimisation
G06F 15/00 - Digital computers in generalData processing equipment in general
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
G06F 19/24 - for machine learning, data mining or biostatistics, e.g. pattern finding, knowledge discovery, rule extraction, correlation, clustering or classification
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.
An embodiment of a method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance is described that comprises the steps of: generating cDNA species from each RNA molecule in an HIV sample population; amplifying at least one first amplicon from the cDNA species, wherein each first amplicon comprises a plurality of amplified copies and is amplified with a pair of nucleic acid primers that define a locus of the first amplicon; clonally amplifying the amplified copies of the first amplicons to produce a plurality of second amplicons wherein a plurality of the second amplicons comprise an immobilized population of substantially identical copies from one of the amplified copies of first amplicons; determining a nucleic acid sequence composition of the substantially identical copies from at least 100 of the immobilized populations in parallel on a single substrate; and detecting one or more sequence variants that occur at a frequency of 5% or less in the nucleic acid sequence composition of the at least 100 immobilized populations; and correlating the detected sequence variants with variation associated with HIV tropism.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
7.
System and method for emulsion breaking and recovery of biological elements
An embodiment of a method for extracting biological material from an emulsion is described that comprises the steps of a) breaking an emulsion comprising a plurality of aqueous droplets in a continuous phase of an oil using a solvent to produce a combined aqueous-oil mixture, where the solvent disrupts the aqueous droplets which release a plurality of biological elements each immobilized on a substrate into the combined aqueous-oil mixture; b) introducing an inorganic salt to the combined aqueous-oil mixture causing a phase separation of the mixture into a first phase comprising an aqueous solution and the biological elements and a second phase comprising the solvent and the oil; c) extracting the first phase from the second phase; and d) collecting the substrate immobilized biological elements from the first phase.
An embodiment of method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises (a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; (b) generating a value for the signal; and (c) correcting the value for the phasic synchrony error using a first parameter and a second carry forward parameter.
G06F 19/00 - Digital computing or data processing equipment or methods, specially adapted for specific applications (specially adapted for specific functions G06F 17/00;data processing systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes G06Q;healthcare informatics G16H)
G06F 15/00 - Digital computers in generalData processing equipment in general
9.
SYSTEM AND METHOD FOR ADAPTIVE REAGENT CONTROL IN NUCLEIC ACID SEQUENCING
An embodiment of a method for adaptive reagent control is described that comprising a) introducing a first concentration of an enzyme reagent into a reaction environment with a reaction substrate, where the enzyme reagent and reaction substrate are constituent parts of a sequencing process; b) measuring a level of activity of the first concentration of the enzyme reagent in the reaction environment, where the level of activity comprises a measurable product of a reaction between the enzyme reagent and the reaction substrate; c) identifying an optimal concentration using the measured level of activity of the first concentration; and d) performing the sequencing process in the reaction environment using the optimal concentration of the enzyme reagent, where the sequencing process comprises an iterative series of sequencing reactions.
An embodiment of an identifier element for identifying an origin of a template nucleic acid molecule is described that comprises a nucleic acid element comprising a sequence composition that enables detection of an introduced error in sequence data generated from the nucleic acid element and correction of the introduced error, where the nucleic acid element is constructed to couple with the end of a template nucleic acid molecule and identifies an origin of the template nucleic acid molecule.
In one embodiment of the invention a method for detecting low frequency occurrence of one or more HIV sequence variants associated with drug resistance is describe that comprises generating cDNA species from RNA molecules in an HIV sample population; amplifying first amplicons from the cDNA species, wherein each amplicon comprises amplified copies and is amplified with a pair of nucleic acid primers that define a locus; clonally amplifying the amplified copies of the first amplicons to produce second amplicons that comprise an immobilized population of substantially identical copies from one of the amplified copies of first amplicons; determining a nucleic acid sequence composition from at least 100 of the immobilized populations in parallel on a single instrument; detecting one or more sequence variants that occur at a frequency of 5% or less in the nucleic acid sequence composition of the at least 100 immobilized populations; and correlating the detected sequence variants with variation associated with HIV drug resistance.
The present invention provides for a method of preparing a target nucleic acid fragments to produce a smaller nucleic acid which comprises the two ends of the target nucleic acid. Specifically, the invention provides cloning and DNA manipulation strategies to isolate the two ends of a large target nucleic acid into a single small DNA construct for rapid cloning, sequencing, or amplification.
An embodiment of method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copie of a template molecule is described that comprises (a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; (b) generating a value for the signal; and (c) correcting the value for the phasic synchrony error using a first parameter and a second carry forward parameter.
G06F 19/22 - for sequence comparison involving nucleotides or amino acids, e.g. homology search, motif or Single-Nucleotide Polymorphism [SNP] discovery or sequence alignment
14.
METHODS OF AMPLIFYING AND SEQUENCING NUCLEIC ACIDS
An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.
G06F 19/00 - Digital computing or data processing equipment or methods, specially adapted for specific applications (specially adapted for specific functions G06F 17/00;data processing systems or methods specially adapted for administrative, commercial, financial, managerial, supervisory or forecasting purposes G06Q;healthcare informatics G16H)
New biochemical protocols for high throughput processing of mRNA samples into cDNA libraries with adaptor sequences compatible with automated sequencing systems are provided. The provided methods produces cDNA libraries which do not have 3' bias 5 associated with current cDNA library production methods. New methods for the production of DNA libraries from DNA are also provided.
An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
17.
Methods of amplifying and sequencing nucleic acids
An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12M 1/40 - Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12Q 1/66 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving luciferase
patents
