AU2014342414B2 - Next generation genomic sequencing methods - Google Patents

Next generation genomic sequencing methods Download PDF

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AU2014342414B2
AU2014342414B2 AU2014342414A AU2014342414A AU2014342414B2 AU 2014342414 B2 AU2014342414 B2 AU 2014342414B2 AU 2014342414 A AU2014342414 A AU 2014342414A AU 2014342414 A AU2014342414 A AU 2014342414A AU 2014342414 B2 AU2014342414 B2 AU 2014342414B2
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Luke T. Daum
Gerald W. Fischer
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Longhorn Vaccines and Diagnostics LLC
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    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

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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.

Description

NEXT GENERATION GENOMIC SEQUENCING METHODS
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SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted 5 in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 7, 2013, is named 3022.019.PCT_SL.txt and is 37,821 bytes in size.
FIELD 10 This disclosure is directed to tools, compositions and methods for identifying genetic mutation and mega-bases of nucleic acid infonnation by sequencing and, in particular, to electronic media and programs for analyzing sequences, genes and complete genomes by sequencing, and to the mutations identified and kits comprising reagents for identifying mutations in biological samples. 15
DEFINITION
In the present description and claims, the term “comprising” shall be understood to have a broad meaning similar to the term “including” and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the 20 exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term “comprising” such as “comprise” and “comprises”.
BACKGROUND
Mycobacterium tuberculosis (MTB), the causative agent for tuberculosis, is a 25 highly transmissible bacterial pathogen with significant morbidity and mortality, particularly in HIV infected patients. Since 1997 tuberculosis has remained the leading cause of death in South Africa, a statistic linked to this country’s growing HIV epidemic. Moreover, effective treatment measures in patients with active MTB have been exacerbated by increasing cases of multidrug resistance (MDR) and extensively 30 drug-resistant (XDR) clinical isolates.
Microscopy remains the cornerstone for diagnosing MTB in many low resource areas of the world where both MTB and also HIV are prevalent. However, many HIV infected patients with MTB are smear negative and microscopy provides no information about antibiotic resistance. The emergence of multidrug-resistant (MDR) 5 and extensively drug-resistant strains (XDR) has rendered standard MTB treatment regimens ineffective. According to one study, approximately 20% of TB patients in South Africa with HIV have MDR MTB. Rapid detection of MTB and initiating effective therapy is critical to decrease transmission and improve treatment outcome. The roll-out of Cephiad’s Gene Xpert (Xpert) has improved MTB diagnosis and 10 provides evidence of Rifampin resistance, but information about other drugs is not provided. Furthermore, it may not be feasible to place Xpert testing in many microscopy labs in low resource settings. The ability to efficiently ship sputum samples centrally for next-generation sequencing (NGS) offers an opportunity to utilize highly trained staff and available infrastructure at central or regional laboratories. ο (N S'
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(N H o (N 15 MDR tuberculosis strains are resistant to the first line antibiotics rifampin (RIF) and isoniazid (INH), while XDR MTB strains are resistant to both RIF and INH as well as any fluoroquinolone and second-line injectable antibiotic drugs (e.g., amikacin, kanamycin or capreomycin). About 6% of all MTB cases are MDR strains and South Africa continues to report higher percentages of XDR cases each year. While 7% of 20 patients infected with standard MTB strains succumb to infection, the death rate rises to almost 50% with MDR tuberculosis. The emergence of antibiotic resistant MTB strains underscores an immediate need for rapid and highly accurate diagnosis, particularly in the developing countries of Africa. In addition migratory populations make geographical surveillance and tracking of drug resistance strains more urgent. 25 Culture-based drug susceptibility testing (DST) for MDR strains is considered the gold-standard, but is time consuming (weeks to months), technically challenging and cost prohibitive, especially in resource limited countries. For example, the BACTEC MGIT 960 (Becton Dickinson Microbiology System, Silver Sparks NV, USA), is an automated continuously culture-based monitoring system that measures 30 bacterial oxygen consumption and can perform DST using prepared kits which are available for susceptibility of strains to a number of antibiotics. DST results obtained with the BACTEC MGIT 960 yield reliable and reproducible but require handling of viable and potentially infectious cultures, days to weeks or delay until results are available, specialized laboratory accommodations and high costs associated with the instrument and consumables.
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(N cn o (N 5 In recent years, several nucleic acid based assays for determining MTB drug resistance have been developed. One of the most popular commercially available diagnostic assays is the GenoType MTBDRplus Line Probe Assay (LPA) by Hain LifeScience. This test employs nucleic acid extraction, PCR amplification, probe hybridization and colorimetric visualization on lateral strips via an alkaline phosphatase 10 reaction. LPA has been shown to be sensitive and specific, but there are several drawbacks. Sensitivity of the LPA for all resistance-associated mutations will most likely never reach 100% since many mutations that confer resistance have yet to be discovered. Another inherent limitation of the LPA is an inability to detect sample populations that contain a mixture of resistant and susceptible strains. Strains that 15 harbor substitution mutations that change an amino acid to a previously uncharacterized or unknown mutation not presented on the LPA are not detected. Furthermore, the LPA only allows detection of the most frequent mutations that cause resistance. If a strain were to contain mutations outside of the targeted mutations, the wild-type banding pattern will appear leading to a false negative (susceptible) result. 20 Thus, there is a need for a rapid, standardized, cost-effective protocol for full length gene analysis of critical genes such as, for example, genes associated with first and second line drug resistance.
The reference to prior art in the background above is not and should not be taken as an acknowledgment or any form of suggestion that the referenced prior art 25 forms part of the common general knowledge in Australia or in any other country.
SUMMARY OF THE DISCLOSURE 30
At least one embodiment in accordance with the disclosure ameliorates the potential shortcomings associated with current strategies and designs, and hereby provides tools, compositions, methods to facilitate and simplify sequencing and r-
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According to one embodiment of the disclosure there is provided a rapid and sensitive method of identifying a nucleic acid sequence motif of an organism 5 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 with multiple primer pairs, wherein each primer pair of the multiple primer pairs comprise primers that are from 15 to 25 nucleic acids in length, have a GC content of about 25%-50%, and an annealing temperature within 10 5°C; obtaining sequence information of the amplified sequences by ne.xt generation sequencing, wherein the amplified sequences represent overlapping portions of the complete sequence of the organism; determining a polymorphism in the genome of at least one strain or serotype from the sequence information obtained; and coiTelating the polymorphism identified with a phenotype or genome location of the at least one strain 15 or serotype to identify the motif.
Preferably, the motif is indicative of the presence of a pathogen. Preferably, the organism is one or more of a virus, a bacterium, a fungus or a parasite. Preferably, the virus is one or more of a DNA virus, an RNA vims, a positive or negative single-strand virus, a double strand virus, an orthomyxovirus, a paramyxovirus, a retrovirus, a 20 flavivirus, a filovirus, a lentivirus, an influenza virus, a human immunodeficiency vims, a hepatitis virus, or an ebola virus. Preferably, the bacterium is Mycobacterium tuberculosis, Plasmodium falciparum, Francisella tularensis, Yersinia pestis, or Vibrio cholera. Preferably, the biological sample is bodily fluid and/or tissue obtained from a patient. Preferably, the motif does not specifically hybridize to other nucleic acid 25 sequences of the organism. Preferably, the samples are provided in a molecular transport medium and the molecular transport medium contains a chaotrope, a detergent, a reducing agent, a chelator, a buffer, and an alcohol, together present in an amount sufficient to lyse cells, denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid. Preferably, the samples are provided at ambient 30 temperatures without refrigeration. Preferably, the next generation sequencing is ion torrent sequencing. Preferably, the ion torrent sequencing is performed in a single
ο (N
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(N H o (N reaction. Preferably, the method further comprises at least two motifs, wherein the presence or absence of the two amplified motifs is determined together. Preferably, the motif contains a region that encodes an antimicrobial gene sequence. Preferably, the antimicrobial gene sequence encodes an antibiotic. Preferably, the polymerase chain 5 reaction is carried out in an aqueous mix comprising: a polymerase and optionally a reverse transcriptase; a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP, a chelating agent, an osmolarity agent, an albumin, a magnesium salt; and a buffer.
One embodiment of the disclosure is directed to analyzing drug resistance 10 mutations by semi-conductor sequencing and, preferably, ion torrent sequencing. Nucleic acid segments containing a gene of interest are amplified by PCR and the amplified products are processed and subsequently analyzed by sequencing. For nucleic acid segments that comprise RNA, the RNA is reverse transcribed to DNA. Sequencing is preferably by Ion Torrent, or Next-Generation sequencers including the 15 Ion Torrent Personal Genome Machine (PGM^M; Life Technologies).
Preferably, the amplification products represent a common full-length, or multiple overlapping pieces of genes of a number of species, strains and/or serotypes of organisms. The amplified products are sequenced and mutations identified and mapped. Mapping identifies both known and previously unknown mutations and is 20 useful to track the progress and movement of drug resistance across a population. Preferably, the disclosure analyzes nucleic acids of pathogens such as, for example, virus, bacteria or parasites. Preferably the viral pathogens are the causative agents of influenza or HIV and the bacterial pathogens are the causative agents of tuberculosis. Ion torrent sequencing of the nucleic acid segments provides enhanced sequencing for 25 rapid, efficient, cost-effective protocol for full length gene analysis. Drug resistance and other mutations are immediately determined.
Another embodiment of the disclosure is directed to tools, compositions and methods for performing NGS sequencing, preferably ion torrent or MiSeq'*’^ sequencing of genes or complete genomes. The disclosure comprises obtaining a DNA 30 sequence of an organism of interest and performing polymerase chain reaction analysis using multiple pairs of nucleic acid primers. Each pair of primers is designed to ο (N S'
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(N m o (N simultaneously amplify overlapping segments of the genome under similar PCR conditions and these may be performed as sequencing reactions or multiplex for multiple genes or the entire genome. Preferred primers possess similar GC content and overall size. A single PCR amplification of the genome produces hundreds of 5 amplification products whose sequences include the full-length gene, large gene and noncoding segments or the entire genome of the organism. These products are analyzed, preferably by NGS, and the sequences matched to create a sequence map of the entire gene or genome.
Another embodiment of the disclosure is directed to methods of identifying a 10 sequence motif in the genome of a microorganism that confers resistance to an antimicrobial compound, comprising: providing multiple nucleic acid samples obtained from multiple different strains or serotypes of the microorganism; amplifying the sequences of the multiple nucleic acid samples by a polymerase chain reaction; obtaining sequence information of the amplified sequences by ion torrent sequencing; 15 identifying a polymorphism in the genome of at least one microorganism strain or serotype from the sequence information obtained; and correlating the polymorphism identified with a phenotype or genome location of the at least one microorganism strain or serotype to identify the sequence motif that confers resistance to the antimicrobial compound. 20 Preferably, the microorganism is a virus, a bacterium, a fungus or a parasite, and the virus is influenza vims and the bacterium is Mycobacterium tuberculosis. Preferably, the nucleic acid samples are provided in an aqueous molecular transport medium that contains a chaotrope, a detergent, a reducing agent, a chelator, a buffer, and an alcohol, together present in an amount sufficient to lyse cells, denature proteins, 25 inactivate nucleases, kill pathogens, and not degrade nucleic acid. Preferably, amplifying is performed in a one step polymerase chain reaction utilizing a primer pair that amplifies a gene or nucleic acid segment associated with resistance to an antimicrobial compound, and the polymerase chain reaction is earned out in an aqueous mix comprising; a heat-stable polymerase; a mix of deoxynucleotide tri phosphates 30 comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP, a chelating ο (N S'
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(N m o (N r- agent, an osmolarity agent, an albumin, a magnesium salt; and a buffer. Preferably the antimicrobial compound is an antibiotic.
Another embodiment of the disclosure is directed to methods of treating a disease or disorder caused by the at least one microorganism strain or serotype with the 5 antimicrobial compound identified by the methods of the disclosure. Preferably, treatment comprises the targeted killing of the specific pathogen that is the causative agent of the disease or disorder. Also preferably, the effective dose is determined from methods of the disclosure by assessing the phenotypic characteristics associated with the target sequence or sequences identified. 10 Another embodiment of the disclosure is directed to methods for determining a complete sequence of a target gene or genome of an microorganism comprising: producing a series of amplicons by performing a single polymerase chain reaction (PCR) of the genome in an aqueous mixture containing a heat-stable polymerase; a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, 15 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 is from 15 to 25 nucleic acids in length, has a GC content of about 25%-50%, and has a similar annealing temperatures; sequencing each of the series of amplicons produced by next generation sequencing in a single reaction wherein the amplicons represent overlapping 20 portions of the complete sequence of the target gene or genome, and correlating the sequences of the amplicons and constructing the complete sequence of the genome.
Preferably, the target gene or genome may be RNA and the target may be reverse transcribed to DNA before PCR. Preferably, each of the primers of the multiple primer pairs comprise primers that are from 15 to 25 nucleic acids in length and each 25 has a GC content of about 25-50%. Preferably, each primer pair is designed to PCR amplify an amplicon, and the collection of amplicons that are PCR amplified encompass overlapping segments of the complete sequence of the target. Preferably, the plurality of primer pairs hybridizes to the target with primer pairs and are spaced along the genome at about every 500 to 2,000 nucleotides. Preferably, the target gene 30 or genome is of an organism and the organism is a virus, a bacterium, a fungus, a parasite or a cell. Preferably, the virus is one or more of a DNA virus, an RNA virus, a
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(N m o (N r- positive or negative single-strand virus, a double strand virus, an orthomyxovirus, a paramyxovirus, a retrovirus, a flavivirus, a filovirus, a lentivirus, an influenza virus, a human immunodeficiency virus, a hepatitis virus, or an ebola virus. Preferably, the bacterium is one or more of Mycobacterium tuberculosis, Plasmodium falciparum, 5 Francisella tularensis, Yersinia pestis, or Vibrio cholera.
Another embodiment of the disclosure is directed to methods for determining the sequence of a nucleic acid target in one cycle of steps comprising; providing a sample containing the nucleic acid; performing a polymerase chain reaction on the nucleic acid of the sample to produce a series of amplicons, wherein the reaction 10 comprises a heat-stable composition comprising: a polymerase; a mix of deoxynucleotide tri phosphates 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 is from 15 to 25 nucleic acids in length and has a GC content of about 25%-50%, and has an annealing 15 temperature within 5°C; sequencing each of the series of amplicons produced by semiconductor sequencing, and correlating the sequences of the amplicons and constructing the sequence of the nucleic acid target.
Preferably, the nucleic acid of the sample may be RNA and the RNA may be reverse transcribed prior to PCR. Preferably the nucleic acid segment is 1 Mb or greater 20 in length, more preferably greater 2 or more Mb in length, more preferably 5 or more Mb in length and more preferably 10 or more Mb in length. Preferably, each of the primers of the multiple primer pairs is of from 16 to 24 nucleotides in length, has a GC content of about 28-35%, and has an annealing temperature of within 3°C of each other primer. Preferably, each primer pair is designed to PCR amplify an amplicon 25 representing a portion of the sequence of the nucleic acid target, and the collection of amplicons that are PCR amplified represent overlapping portions of the complete sequence of the target. Preferably, the plurality of primer pairs hybridizes to the segment at a spacing of about 800 to 1,500 nucleotides in length.
Another embodiment of the disclosure is directed to mixtures comprising 30 multiple pairs of nucleic acid primers wherein each primer is from 15 to 25 nucleic acids in length and has a GC content of about 25%-50%, and wherein, upon subjecting ο (N S'
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(N m o (N the collection to a polymerase chain reaction in association with a nucleic acid segment, the collection of primer pairs generates a collection of amplicons, wherein each amplicon is about 500 to 2,000 nucleotides in length, such that the entire sequence of the segment is represented in the resulting collection of amplicons. 5 Preferably, each primer of the collection of primer pairs has a GC content of about 25-45%, and an annealing temperature within 3°C of each other primer, and each primer of the collection of primer pairs contains a sequence that hybridizes to the genome of the same microorganism. Preferably, the target may be a gene or genome of a microorganism. Preferably, the gene may be a gene that confers antibiotic resistance 10 of the microorganism. Preferably, the microorganism is a virus, a bacterium, a parasite, or a fungus. Preferably, the mixture contains a heat-stable polymerase; a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP; a chelating agent; a salt; a buffer; a stabilizing agent and nuclease-free water. Preferably, the nucleic acid primers may not self-hybridize. Also preferably, 15 the primer pairs to two or more genes may be multiplexed together.
Another embodiment of the disclosure comprises kits containing reagent vessels preferably including one or more of chemical reagents, primers and polymerases for sequencing. The sample to be analyzed is mixed with a reagent vessel that preferably contains chemical components sufficient to kill all pathogens present in the sample, 20 inactivate nucleases in the sample, and maintain the integrity of the nucleic acids rendering the sample safe for transportation and subsequent manipulation, such as, for example, aqueous lysis buffer, aqueous or anhydrous transport medium, or aqueous PrimeStore Molecular Transport Medium®.
The mixture may be combined in a column, such as a micro-centrifuge column, 25 which may be included in the kit, to aid in the extraction of nucleic acid form the sample. Extracted nucleic acid is preferably combined with another chemical reagent composition such as, for example PrimeMix® that facilitates nucleic acid testing such as, for example, PCR sequencing. Such reagent composition may contain positive control sequences, negative control sequences and/or sequences that specifically 30 hybridize (under the desired high or low stringency hybridization conditions) to a particular target sequences that is characteristic for the presence of a pathogen. ο (N a
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Another embodiment of the disclosure is directed to computer-readable media that implements the analytical methods of the disclosure.
Preferable the computer-readable media analyses sequence information obtained and centralizes the collection of information. Also preferably the sequence information is compared with sequence information obtained from one or more known databases of sequence information for the same or similar sequences and identifies mutations that provide antibiotic resistance and other phenotypic characteristics to the microorganism.
Other embodiments and advantages of the disclosure are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Illustrates the pncA gene sequence plus 100 flanking base pairs as well 15 as the reverse compliment sequence, the protein sequence, and the primers sequences. Figure 2 Illustrates the nucleotide sequence of H37RV Gene strain as well as the sequences of the TB 16S ribosomal RNA gene sequencing primers.
Figure 3 Illustrates the rpoB gene conferring sensitivities/resistance to Rifampin as well as the forward and reverse primer sequences for rpoB. 20 Figure 4 Illustrates the Mycobacterium tuberculosis H37Ra, complete genome (GenBank: CP000611.1) GyrA Gene and three sets of forward and reverse primers. Figure 5 Mycobacterium tuberculosis H37Ra, complete genome (GenBank: CP000611.1) catalase-peroxidase-peroxynitritase T katG and three sets of forward and reverse primers. 25 Figure 6 Illustrates the cycle threshold of Gyrase A and IS 6110 assays.
Figure 7 Illustrates Gyrase A assay and the IS 6110 assay using sequence isolates by cycle number vs. Ct value.
Figure 8 Illustrates Gyrase A assay and the IS 6110 assay using sequence isolates vs cycle threshold (ct). 30 Figure 9 Summary of results achieved in sequencing the influenza A genome using various primer pair collections with ion torrent sequencing methodology. 10
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Figure 10 Characterization of primer pairs for whole-genome ion torrent sequencing of influenza A (H3N2).
Figure 11 (A) Gene sequence of pncA showing coding regions as shaded, and (B) pncA forward and reverse primers utilized in PCR tiling and pncA regions P1-P4. Figure 12 Architecture of an electronic system of the methods of the disclosure.
DETAILED DESCRIPTION
Rapid analysis of genes associated with drag resistant strains is a major challenge for successful treatment of many diseases and disorders. Real-time 10 geographical surveillance of emerging MTB drug resistance would facilitate more appropriate treatment strategies (e.g., drug, antibiotic, chemical). Currently, available molecular methods such as the GenoType® MTBDR/j/m,!» LPA offer limited detection capabilities, particularly when novel/uncommon amino acid substitutions are within known drug resistance regions or when undiscovered amino acid mutations impact drug 15 resistance. Also, cuiTent methodology including Ion Torrent protocol requires multisteps, ancillary equipment and increased expense, and is labor intensive. A simplified semiconductor sequencing protocol for rapid characterization of full-length genes and genome has been surprisingly discovered. The disclosure comprises a standardized protocol for gene sequencing preferably utilizing 20 semiconductor sequencing and preferably Ion Torrent sequencing of full-length genes. The protocol enables sequencing of entire coding regions implemented allowing characterization of known mutations and discovery of new polymorphisms. This protocol also enables the sequencing of mega-bases of nucleotide information such that complete genomes of cells and organisms can be determined and the genetic 25 polymorphism readily mapped and identified. Preferably the cells or organisms are disease causing prokaryotic or eukaryotic cells, or yeast or fungal cells. Preferred disease causing organisms include strains of bacteria, virus, fungus, and parasites. Exemplary organisms include, but are not limited to DNA virus, an RNA virus, a positive or negative single-strand virus, a double strand virus, orthomyxovirus, 30 paramyxovirus, Morbillivirus (e.g., Rubeola), retrovirus, flaviviras, filovirus, lentivirus, hanta virus, herpes virus (e.g., VZV, HSV I, HSV II, EBV), hepatitis virus (e.g., A, B, 11 ο (Ν (Ν (Ν m ο (Ν C, ηοη-Α, ηοη-Β), Influenza virus (e.g., Η5Ν1, MINI, H7N9), Respiratory Syncytial Virus, HIV, or Ebola virus. Exemplary organisms also include but are not limited to Mycobacteria (e.g., M. tuberculosis), Bacillus anthracis, Plasmodium (e.g.. Plasmodium falciparum), Shistosomiasis (e.g.. Schistosoma mansoni), Francisella 5 tularensis, Clostridium difficile. Meningococcal infections. Pseudomonas infections. Yersinia pestis, and Vibrio cholerae. The disclosure is also directed to the detection and characterization of organisms that are related to the pathogenic organisms, but are non-pathogenic. Detection of one or more of the non-pathogenic, but related organisms can be a definitive diagnosis of the absence of disease. In addition, the tools and 10 methods of the disclosure allow for the identification and characterization of abnormalities in the existing genome of an individual such as a condition that may be present from birth (congenital) and may be heritable. These genetic disorders are equally detectable and characterizable by the tools and methods of the disclosure and can be diagnosed by comparison with an otherwise normal or control genome of a non-15 afflicted individual.
This relatively rapid (e.g., 1, 2, or 3 days, or less), standardized, cost-effective protocol allows for full-length analysis of genes such as, for example, to identify mutations that possess one or more alterations of a DNA, RNA, protein and/or peptide sequence. For sample sequences that are RNA, the RNA sequence of interest in the 20 sample is typically reverse transcribed to DNA for PCR analysis. Preferably identified and characterized are one or more gene mutations that provide a microorganism with resistance to an antibiotic. Preferred mutations that are identified with the methods of the disclosure are located in one or more sites within an amino acid coding region, a transcription promoter or termination site, a stop or start codon, a site within a non-25 coding region, a splice junction site, a modification site, a transcription or translation factor binding or recognition site, one or more sites that contribute to a three dimensional structure, or a combination thereof. Preferred genes that are analyzed include MTB genes associated with first and second-line MTB drug resistance (see Figures 1-5). Preferred examples of MTB-associated genes include, for example, rpoB 30 (rifampin), katG and inhA (isoniazid), gyrA and gyrB (fluoroquinolones), pncA and 12 ο (Ν δ' (Ν (Ν Η Ο (Ν ο panD (ΡΖΑ or pyrazinamide) and rrs(16s) (aminoglycosides, amikacin, kanamycin, capreomycin, streptomycin) and rspL (streptomycin).
The methods of the disclosure were used to evaluate 26 geographically diverse clinical isolates collected in South Africa including MDR and XDR strains with next-5 generation Ion Torrent Personal Genome Machine (PGM). Of particular interest were INDELS, which are insertions or deletions if a single nucleotide (A,T,G,C) causing missense changes in the protein structure. The sequencing data obtained from this developed methodology were compared to the HAIN LPA and genotyping DST data from culture. This methodology for the first time enables sequencing entire coding as 10 well as non-coding regions for genes implemented in resistance allowing characterization of known mutations and discovery of new polymorphisms. Previously uncharacterized substitution mutations were identified on the its, rpoB, katG, pncA gyrA and gyr B, katG, inhA and panD genes.
The present disclosure offers significant potential for new sequencing platforms 15 such as, for example, next-generation instruments to be more utilized in resource deprived environments such as Africa, Asia, and India. Specifically, the current disclosure improves and streamlines the up-front library preparation process.
Methodology of the disclosure does not require the use of expensive ancillary equipment pieces typically utilized or required by the manufacturer. Specifically, the 20 standardized procedure of the disclosure does not require an Agilient Bioanalyzer for DNA quantifications; the OneTouch ePCR system for emulsification PCR step, or the PipinPrep for gel excision. Additionally, since the protocol of the disclosure involves re-sequencing full-coding genes (not necessarily full genomes) the Bioruptor is not required for shearing DNA into smaller pieces. Additionally, it is not necessary to 25 sequence the entire genome and then identify genes. The method and tools of the disclosure allow for pre-selection of the genes and/or regions of interest that are to be sequenced. As the Agilent 2100 BioAnalyzer, OneTouch, PipinPrep, and Bioruptor all require additional training for use, consume valuable laboratory bench space, and are extremely expensive, the disclosure represents a significant advance and improvement 30 over convention methodologies. 13 ο (Ν (Ν (Ν m ο (Ν ο
The sequencing protocol of the disclosure is exemplified herein using Ion Torrent sequencing as this sequencing method has been applied to M. tuberculosis. As believed to clear to those skilled in the art, the protocol involves semiconductor sequencing, with is exemplified by Ion Torrent sequencing and, as such, involves the 5 sequencing of large numbers of different regions simultaneously. The sequencing and nucleic acid methodologies are applicable to any series of genes, genomes or nucleic acid sequences.
The disclosure also includes a methodology for selecting primer pairs for sequencing a target of interest. Primer pairs are preferably selected with matched 10 annealing and melting temperature as to the target. Preferably, melting and annealing temperatures are based on sequence characteristics such as the GC content of the sequence, the possibility of self-hybridization of the primer (e.g., forming hairpin loops within the primer), and possible structures near the binding site. Preferably the primers do not self-hybridize under the conditions of sequencing. Preferably the GC content of 15 primers is between about 25% and 50%, more preferably between about 30% and 40%, more preferably between about 25% and 35%, and also more preferably between about 40% and 50%. Thus, primer sequences of the target are selected for hybridization based on sequence characteristics such that all of the primer pairs utilized for the target will have similar melting and/or annealing temperatures to the target. Preferably 20 primer sequences contain no regions of reasonably possible self hybridization of the primer sequence. Preferably primer pairs are matched for annealing and/or disassociation temperatures which may be within 5°C, within 4°C, with 3°C, within 2°C, with 1°C and more preferably the same annealing temperature, the same melting temperature or both. Primer pairs preferably generate amplicons of between about 500 25 to about 2,000 nucleotides (NT) in length that represent overlapping segment of the target, more preferably between about 600 and 1,500 NT, more preferably between about 700 and 1,300 NT, more preferably between about 800 and 1,200 NT, more preferably between about 900 and 1,100 NT, and more preferably about 1,000 NT. Primers are generally between 12 NT and 45 NT in length, more preferably between 15 30 and 35 NT, and more preferably between about 18 and 25 NT. Although not a rule, generally longer primers have a lower GC content. Exemplary primers pairs are 14 ο (Ν δ' (Ν (Ν m ο (Ν ο identified for the pncA gene (see Figure 1), the H37RV gene strain (see Figure 2), the rpoB gene (see Figure 3), the GyrA gene (see Figure 4, and the katG gene (see Figure 5). These primer pairs are useful to combine in ready to use kits to simplify the sequencing of full-length genes. 5 In one embodiment of the disclosure, a semiconductor sequencing protocol was determined for five genes of M. tuberculosis for determining drug resistance in MDR and XDR strains (e.g., cumulatively sequencing 11.4 kb per isolate). The M. tuberculosis rpoB gene encodes a 1,178 amino acid beta subunit for an RNA dependent DNA polymerase enzyme. Mutations within an 81-bp “core region” of the ψοΒ gene 10 are responsible for approximately 95% of rifampin resistance in M. tuberculosis strains. Three of these mutations at positions 516 (D^V), 526 (H^Y/D), and 531 (S—>L) constitute the majority of mutations within this region. Of the 21 rifampin-resistant strains characterized in this study, 11 (52.4%) carried the S531L mutation, 7 (33.3%) contained an amino acid substitution at position 516, and 3 (14.3%) contained a 15 mutation at position 526 of the rpoB gene (Table 1). The most prevalent rpoB substitution observed at position 516 is a valine (D516V). Ion Torrent sequencing according to the disclosure revealed that 6 of 7 strains contained a rarer glycine residue (D516G) at this position (Table 1). These 6 strains were shown as absent for both mutant and wild type bands by LPA (Table 1). Similarly an uncommon amino acid 20 substitution was identified at position 526 in the rpoB gene. The most prevalent amino acid substitution reported at position 526 in the rpoB gene is a histidine to tyrosine or aspartic acid (H526Y/D). Ion Torrent sequencing revealed 1 of 3 isolates contained an uncommon arginine (R) residue (H526R) that by HAIN LPA was shown to be absent for both wild type and mutant bands (Table 1). While absence of wild type and mutant 25 bands in a sample are interpreted as resistant according to LPA testing, there remains ambiguity since the type of amino acid change is not directly characterized. This underscores the utility of Ion Torrent sequencing for resistance surveillance, and discovery of novel amino acids in circulating MTB strains.
Table 1 30 Summary of 10 amino acid mutations in the first 900 amino acid residues* of the rpoB gene of 26 (14 MDR, 7 XDR and 5 fully susceptible) M. tuberculosis isolates 15 20 25
ο (NS'(N
(N mo (N 15 10 from South Africa deduced by Ion Torrent sequencing, Hain LPA genotyping and culture. Isolate No. Amino Acid Substitution(s)** Rifampin Result by of ruoB sene (3619 bosi Ion Torrent* MAIN LPA Bacter MGIT 960 9 S531L Resistant Resistant Resistant 1 S531L, V1941 Resistant Resistant Resistant 1 S531L, Y645H Resistant Resistant Resistant 1 H526D Resistant Resistant Resistant 1 H526Y, S509R Resistant Resistant Resistant 1 H526R Resistant Resistant Resistant 5 wild type ** Sensitive Sensitive Sensitive 6 D516G, L533P Resistant Resistant Resistant 1 D516V Resistant Resistant Resistant * There were 5 rpoB amino acid substitutions (R908C, Q1042H, P1043A, I1187T, and V1249F) noted in at least 1 strain at the 3' end {residues 900-1253). ** Compared to the sequenced H37Rv reference strain. 30 40
The katG gene encodes catalase peroxidase, an enzyme that converts isoniazid (INH) into the active form. The majority of isoniazid resistance is associated with katG codon 315 (S315T), although mutations in the promoter region of inhA and nod also contribute to resistance. Of 26 strains assessed, 16 (62%) contained the characteristic serine-to-threonine amino acid substitution at position 315 (S315T) conferring isoniazid resistance (Table 2). These sequencing results exhibited 100% concordance with comparisons made using the HAIN LPA and culture DST.
Table 2 Summary of 4 amino acid mutations in the katG gene of 26 (14 MDR, 7 XDR and 5 fully susceptible) M. tuberculosis isolates from South Africa deduced by Ion Torrent sequencing, Hain LPA genotyping and culture 35 Isolate No. Amino Acid Siibstitution(s)** of katG sene (2447 bus ) Ion Torrent Rifampin Result by * HAIN LPA Bacter MGIT 11 S315T Resistant Resistant Resistant 5 S315T. R463L Resistant Resi.stant Resistant 1 W191R, R463L Sensitive Sensitive Sensitive 7 wild type** Sensitive Sensitive Sensitive 1 R463L Sensitive Sensitive Sensitive 1 NOSH Sensitive Sensitive Sensitive * Rifampin resistance is known to occur in rpoB at positions 531(S^T), 526 (H^Y/D), and 516 (D^V). ** Compared to the sequenced H37Rv reference strain. 45
Pyrazinamide (PZA) is a synthetic derivative of nicotinamide that has been used as a first-line drug to fight tuberculosis since 1952. Standard DST for PZA is 16 35 20 25 Isolate No. Amino Acid Substitution(s)** Pyrazinamide Result by in the nncA gene (3619 bps) Ion Torrent* Bacter MGIT 960 3 C14R Resistant Resistant 1 A102V Resistant Resistant 1 Q122 (stop) Resistant Resistant 16 wild type ** Sensitive Sensitive 1 V139G Resistant Resistant 1 R154G Resistant Resistant 2 L172P Resistant Resistant 1 Silent (C195T) Sensitive Sensitive * pyrazinamide resistance is known to occur in 25 mutations described by Mphahele et al (23). ** Compared to the sequenced H37Rv reference strain. One strain contained a silent (synonymous) nucleotide mutation at position 195 (C—>-T). r-·
ο (N
(N
(No (N 10 15 complicated due to an acidic pH requirement in vitro, which inhibits M. tuberculosis growth and complicates accurate phenotypic assessment. PZA resistance is attributed to mutations in the pncA gene which encodes a pyrazinamidase. These resistance conferring mutations are numerous and include amino acid substitutions, frameshifts and stop codon mutations. Seven mutations were characterized from the 26 South African isolates assessed, including one silent mutation, 5 amino acid substitutions, and one chain termination mutation. The Q122 (Stop) termination mutation (Table 3) observed in one isolate is novel, having not been reported elsewhere. The difficulty in PZA phenotypic assessment and the variability of mutations along the pncA gene further underscores the added value of Ion Torrent gene sequencing to assess mutations in this hyper variable MTB gene.Table 3 Summary of 6 amino acid mutations in the pncA gene of 26 (14 MDR, 7 XDR and 5 fully susceptible) M. tuberculosis from South Africa deduced by Ion Torrent sequencing and culture 30
The primary target of fluoroquinolones (FQ) in M. tuberculosis is DNA gyrase, a type II topoisomerase composed of two A and B subunits encoded by the gyrA and gyrB genes, respectively. Amino acid substitutions located within a short region of the gyrA gene known as the quinolone resistance-determining region (QRDR), account for the majority of known FQ resistant tuberculosis strains. Substitution mutations in the QRDR at positions 88, 90, and 94 were observed in 10 of 26 (38.5%) sequences from this study (Table 4). Three of these 10 strains contained substitutions at position 94 in the gyrA gene; two were noted as D94G substitutions, and one was a D94Y 17
ο (N
Ό (N
(N m o (N 10 25 substitution. Both D94G and D94Y have been characterized as substitutions and both amino acid substitutions at codon 94 give rise to similar levels of FQ antibiotic resistance. Of the strains assessed, the gyrA gene was the most variable containing nine amino acid substitutions in the 26 clinical isolates assessed. Furthermore, two of these gyrA codons (549 and 613), exhibited heterogeneous residues (Table 4), an advantage of performing Ion Torrent sequencing over MAIN LPA and DST.
Table 4
Summary of 10 amino acid mutations in the gyrA gene of 26 (14 MDR, 7 XDR and 5 fully susceptible) M, tuberculosis isolates from South Africa deduced by Ion
Torrent sequencing and culture 15 20
Isolate No. Amino Acid Substitution(s)** Rifampin Result by in the svrA aene (2664 bps) Ion Torrent* Bacter MGIT 960 3 E21Q, S95T, G2475S, G668D Sensitive Sensitive 2 E21Q, D94G, S95T, G668D Resistant Resistant 1 E21Q, G88C, S95T, G668D Resistant Resistant 10 E21Q, S95T, G668D Sensitive Sensitive 1 wild type** Sensitive Sensitive 1 E21Q, S95T, G668D, Q6I3Q/E+ Sensitive Sensitive 1 E21Q, S95T, G668D, L5495/L+ Sensitive Sensitive 1 E21Q, D94Y, S95T, G668D Resistant Resistant 6 E21Q, A90V, S95T, G247S, G668D Resistant Resistant * Fiuroquinolone resistance is known to occur in gyrA at position 88(G^C), 90 (A^V), 91 (S91P)and94(D^H). ** Compared to the sequenced H37Rv reference strain. + There is a heterozygous nucleotide mutation in a proportion of Ion Torrent reads; the mutation confers a mixed amino acid substitution. 30 35 40
Emerging cases of XDR tuberculosis defined as MDR cases having acquired additional resistance to FQ, i.e., ofloxacin, and at least one of the three injectable ‘second-line drugs’, i.e., amikacin (AMK), kanamycin (KAN), or capreomycin (CAP), have become a public health threat in developing countries worldwide. The majority of resistance to second line drugs is associated with mutations in codons 1401 (A1401G), 1402 (C1402T), and 1484 (G1483T) in the 16 S ribosomal RNA rrs gene. Analysis of African MTB strains revealed that 7 of 26 (27%) were defined as XDR as evident by nucleotide mutation at position 1401 (A1401G) (Table 4). Three additional nucleotide mutations at positions 492, 514, and 878 were also discovered (Table 5) in strains from this analysis. The G878A is a novel nucleotide mutation but was shown to be sensitive to AMK, KAN, and CAP according to DST.
Table 5 18 ο (N σ3
Summary of 4 nucleotide mutations in the rrs (16s) gene of 26 (14 MDR, 7 XDR and 5 fully susceptible) M. tuberculosis isolates from South Africa deduced by Ion Torrent sequencing and culture.
(N
(N mo (N 15 20 25 30 35 10 Isolate No. Amino Acid Substitution(s)** Kanamycin Result by in the rrs tl6s) «ene (1680 bns) Ion Torrent* BacterMGIT960 1 G878A Sensitive Sensitive 12 wild type** Sensitive Sensitive 1 A514C, A1401G Resistant Resistant 6 A1401G Resistant Resistant 3 A514C Sensitive Sensitive 1 C492T Sensitive Sensitive 1 C492T, A514C Sensitive Sensitive 1 A5I4C Sensitive Sensitive * Aminoglycoside resistance is known to occur at positions 1401 (A^G), 1402 (C^T), and 1484(G^T). ** Compared to the sequenced H37Rv reference strain. Previous studies have shown that mutations in katG codon 463, and gyrA codon 95 are genetic markers for categorizing strains into epidemiological genetic Groups 1, 2, and 3, and that these codons have no effect on antibiotic resistance. Group 1 strains are genetic ancestors of Group 2 and Group 3 strains that link the predominately nonhuman mycobacterium genus (M. microti and M. bovis strains) with human M. cifricanum and M. tuberculosis lineages. As evident by substitution mutations in katG codon 463 and gyrA codon 95, a total of 7 of 26 (27%), 18 of 26 (69%), and 1 of 26 (4%) of the African isolates chiu-acterized in this study were members of genetic Group 1, 2, and 3, respectively. Tracking Group 1 organisms is important in terms of MTB detection since several isolates belonging to genetic Group 1 lack Insertion Sequence 1661 (IS-1661), a common genetic target for several PCR-based MTB detection assays. The Ion Torrent protocol for MTB drug resistance can be easily integrated into low resource settings throughout countries tuid regions such as Africa, India, and China. The Ion Torrent methodology does not require the use of expensive ancillary equipment such as Agilent 2100 BioAnalyzer, DiaGenode Bioruptor® Sonication System, Ion OneTouch System™, ultracentrifuges, or the Pippin Prep™ Workstation as current Ion Torrent protocols recommend. This is significant since these instruments and needed accessories and consumables can be expensive, require large laboratory footprints, and often require routine maintenance. 19
ο (N
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In contrast to the GenoType® MTBDRp/Μ,ϊ or MTBDRsl Line Probe Assay (LPA), the Ion Torrent PGM protocol provides full-length characterization of genes, making possible discovery of new amino acid substitutions that could potentially be missed by LPA since LPA is limited to only known mutations. Using the protocol, 5 several uncommon amino acid changes in clinical field isolates have been found. Furthermore, the extensive depth of sequence coverage from the Ion Torrent allows for discovery of heterogeneous or mixed strain genetic populations within an isolate.
The scalability of Ion Torrent sequencing permits expansion to include megabases of additional genes on a single chip. The methodology of the disclosure is 10 expandable beyond the five full-length MTB genes to include all 16 plus genes that currently constitute MTB drug resistance. Full-length gene analysis using the Ion Torrent PGM will identify novel mutations that, when correlated to phenotypic minimal inhibitory concentration (MIC) testing, identify new tuberculosis resistant residues as well as the cumulative inhibitory effect of multiple mutations. 15 Another embodiment of the disclosure is directed to megabase sequence identification utilizing semiconductor sequencing protocols. Megabase sequencing according to the disclosure involves selection of primer pairs that amplify different sections of the target sequence whereby the collection of sections represent the entirety of the target sequence. Preferably the sections overlap to a degree that permits 20 alignment of the resulting amplicons forming the complete target sequence. Primer pairs are preferably designed to form amplicons with lengths of about 0.5k to about 5k nucleotides, preferably about 0.6k to about 3 k nucleotides, more preferably about 0.7k to about 2k nucleotides, and more preferably about 0.8k to about Ik nucleotides. Primer pairs are preferably of similar GC contact such that the annealing or 25 hybridization temperatures are as similar or preferably within about 5°C, more preferably within about 2°C, and more preferably within about 1°C. Also preferred is that the hybridization disassociation temperatures be similar, such that annealing and disassociation occur at very similar temperature for polymerization and PCR. In annealing and disassociation, the length of the primer influences the temperature 30 profile, thus similar length for the all or at least most of the primers is preferred. Primer lengths are preferably about 15-30 nucleotides, more preferably about 20-28 20
ο (N
(N
(N m o (N nucleotides, and more preferably about 18 to 25 nucleotides. Although it is preferred that all of the primers have such similar characteristics, megabase sequencing can be performed when greater than about 80% of the primers share one or more characteristics, more preferably 85% or more, more preferably 90% or more, and even 5 more preferably 95% or more. Primer pairs can be assembled into kits to facilitate full-length sequencing. Primers targeted to amplify a target sequence are added to nucleic acid obtained from samples. In accordance with the utilization of such similar primers, a PCR reaction is performed with one target nucleic acid to be amplified with a mixture of all primer pairs. Also preferred is performance of duplicate PCR analysis on 10 identical mixtures. The number of cycles can range from 10 to 50 or more and, preferably temperature cycling is performed in accordance with convention PCR temperature and reaction conditions.
Another embodiment of the disclosure is directed to methods of treating a disease or disorder caused by the at least one microorganism strain or serotype with the 15 antimicrobial compound identified by the methods of the disclosure. Preferably, treatment comprises the targeted killing of the specific pathogen that is the causative agent of the disease or disorder. Also preferably, the effective dose is determined from methods of the disclosure by assessing the phenotypic characteristics associated with the target sequence or sequences identified, and thereby selected known or testing 20 suspected agents for treatment. Preferably, the therapeutically effective dose can be determined from the sequencing information obtained by the sequencing methods of the disclosure. For example, certain sequences, if determined to be present, are known to cause certain phenotypic characteristics, such as, for example, resistance or sensitivity to certain antibiotics or other therapeutic treatments. The presence or 25 absence of these sequences, as well as the quantity of sequences present, can provide an indication and direction of effective treatment as well as the therapeutically effective dose for treatment.
Another embodiment of the disclosure comprises kits containing reagent vessels preferably including one or more of chemical reagents, primers and polymerases for 30 sequencing. The sample to be analyzed is mixed with a reagent vessel that preferably contains chemical components sufficient to kill all pathogens present in the sample. 21 ο (Ν S' (Ν (Ν m ο (Ν inactivate nucleases in the sample, and maintain the integrity of the nucleic acids rendering the sample safe for transportation and subsequent manipulation, such as, for example, aqueous lysis buffer, aqueous or anhydrous transport medium, or aqueous PrimeStore Molecular Transport Medium® (described in U.S. Patent Nos. 8,084,443, 5 8,080,645 and 8,097,419, all of which are specifically incorporated by reference). The mixture may be combined in a column, such as a micro-centrifuge column, which may be included in the kit, to aid in the extraction of nucleic acid form the sample. Extracted nucleic acid is preferably combined with another chemical reagent composition such as, for example PrimeMix® (also described in U.S. Patent 10 Publication No. 2011/0281754 entitled “Compositions and Methods for Detecting, Identifying and Quantitating Mycobacterial-Specific Nucleic Acids” filed April 25, 2011, and hitemational Application Publication No. WO2012/149188 entitled “Compositions and Methods for Detecting and Identifying Nucleic Acid Sequences in Biological Samples” filed April 26, 2012, which are both specifically incorporated by 15 reference), that facilitates nucleic acid testing such as, for example, PCR sequencing. Such reagent composition may contain positive control sequences, negative control sequences and/or sequences that specifically hybridize (under the desired high or low stringency hybridization conditions) to a particular target sequences that is characteristic for the presence of a pathogen. 20 Anotlier embodiment of the disclosure is directed to computer readable programming that implements the methods of the disclosure (see Figure 12). Preferably the computer readable media includes provides formats for including both specific and general information with regard to each sample.. That information can be easily centralized and stored. An exemplary electronic system of the method of the 25 disclosure includes at least one general-purpose computing device 100, including a processing unit (CPU) 120 and a system bus 110 that couples various system components including the system memory such as read only memory (ROM) 140 and random access memory (RAM) 150 to the processing unit 120. Preferably, additional system memory 130 is also available for use. The electronic method may operate on a 30 computing device with more than one CPU 120 or on a group or cluster of computing devices networked together to provide greater processing capability. The system bus 22 ο (N S'
(N
(N m o (N 110 may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures. A basic input/output (BIOS) stored in ROM 140 or the like, may provide the basic routine that helps to transfer information between elements within the computing 5 device 100, such as during start-up. The computing device 100 further includes storage devices such as a hard disk drive 160, a magnetic disk drive, an optical disk drive, tape drive or the like. The storage device 160 is connected to the system hus 110 by a drive interface. The drives and the associated computer readable media provide nonvolatile storage of computer readable instructions, data structures, program modules and other 10 data for the computing device 100. The basic components are known to those of skill in the art and appropriate variations are contemplated depending on the type of device, such as whether the device is a small, handheld computing device, a desktop computer, a computer server, a handheld scanning device, or a wireless devices, including wireless Personal Digital Assistants ("PDAs"), tablet devices, wireless web-enabled or 15 “smart” phones. Preferably, the system is technology agnostic.
Although the exemplary environment described herein employs the hard disk, other types of computer-readable media that can store data that are accessible by a computer, such as magnetic cassettes, flash memory cards, digital versatile disks, cartridges, random access memories (RAMs), read only memory (ROM), a cable or 20 wireless signal containing a bit stream and the like, may also be used in the exemplary operating environment.
To enable user interaction with the computing device 100, an input device 190 represents any number of input mechanisms, such as a microphone for speech, a touch-sensitive screen for gesture or graphical input, keyboard, mouse, motion input, speech, 25 game console controller, TV remote and so forth. The output device 170 can be one or more of a number of output mechanisms known to those of skill in the art, for example, printers, monitors, projectors, speakers, and plotters. In some embodiments, the output can be via a network interface, for example uploading to a website, emailing, attached to or placed within other electronic files, and sending an SMS or MMS message. In 30 some instances, multimodal systems enable a user to provide multiple types of input to communicate with the computing device 100. The communications interface 180 23 ο (Ν (Ν (Ν m ο (Ν ο generally governs and manages the user input and system output. There is no restriction on the disclosure operating on any particular hardware arrangement and therefore the basic features here may easily be substituted for improved hardware or firmware arrangements as they are developed. 5 For clarity of explanation, the illustrative system embodiment is presented as comprising individual functional blocks (including functional blocks labeled as a "processor”). The functions these blocks represent may be provided through the use of either shared or dedicated hardware, including, but not limited to, hardware capable of executing software. For example the functions of one or more processors presented in 10 FIG. 1 may be provided by a single shared processor or multiple processors. (Use of the term "processor" should not be construed to refer exclusively to hardware capable of executing software.) Illustiative embodiments may comprise microprocessor and/or digital signal processor (DSP) hardware, read-only memory (ROM) for storing software performing the operations discussed below, and random access memory 15 (RAM) for storing results. Very large scale integration (VLSI) hardware embodiments, as well as custom VLSI circuitry in combination with a general purpose DSP circuit, may also be provided.
Embodiments within the scope of the present disclosure may also include computer-readable media for carrying or having computer-executable instructions or 20 data structures stored thereon. Such computer-readable media can be any available media that can be accessed by a general purpose or special purpose computer. By way of example, and not limitation, such computer-readable media can comprise RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to carry or 25 store desired program code means in the form of computer-executable instructions or data structures. When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or combination thereof) to a computer, the computer properly views the connection as a computer-readable medium. Thus, any such connection is properly termed a computer-readable medium. 30 Combinations of the above should also be included within the scope of the computer-readable media. 24
ο (N
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Computer-executable instructions include, for example, instructions and data which cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions. Computer-executable instructions also include program modules that are executed by computers 5 in stand-alone or network environments. Generally, program modules include routines, programs, objects, components, and data structures, etc. that performs particular tasks or implement particular abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of the program code means for executing steps of the methods disclosed herein. The particular 10 sequence of such executable insrtuctions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps.
Preferred embodiments of the disclosure may be practiced in network computing environments with many types of computer system configurations, including personal computers, hand-held devices, multi-processor systems, 15 microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, and the like. Networks may include the Internet, one or more Local Area Networks ("LANs"), one or more Metropolitan Area Networks ("MANs"), one or more Wide Area Networks ("WANs"), one or more Intranets, etc. Embodiments may also be practiced in distributed computing 20 environments where tasks are performed by local and remote processing devices that are linked (either by hardwired links, wireless links, or by a combination thereof) through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.
Preferably, the computer-readable media is connected to the Internet and can 25 access publically available databases, such as for example, PubMed or GeneBank and retrieve sequence and related information regarding the microorganism being analyzed including the DNA, RNA and/or protein sequence of one or more genes or portions of genes of the microorganism. The sequences being analyzed by, for example. Ion Torrent sequencing is compared with one or more (e.g., 1, 10\ 10^, 10·^, lO"^, 10^, 10^, 30 10^, or even greater numbers) known sequences of the same or similar microorganism or other synthetic or recombinant sequences. Results achieved can provide a rapid and 25
ο (N
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(N m o (N r- thorough analysis of the gene or gene portion as compared with dozens, hundreds or even thousands of known sequences. Mutations that represent resistance can be easily and rapidly determined and identified.
The following examples illustrate embodiments of the disclosure, but should not 5 be viewed as limiting the scope of the disclosure.
Examples
Clinical Isolates. A total of 26 geographically diverse clinical isolates, representing drug-sensitive, MD, and XDR tuberculosis strains were obtained from sample archives of the University of Pretoria, South Africa, and the National Institute for 10 Communicable Diseases (NICD), Sandringham, South Africa. The H37Rv MTB lab strain was included as a sequencing control throughout the protocol. All MTB isolates used were archived strains from pure culture MGIT™ 960 System tubes (Becton Dickinson, Sparks, MD) with species identification and genotypic resistance to rifampin and isoniazid determined using the Genotype® MTBDp/iis assay (HAIN 15 LifeSciences, Germany) according to manufacturer’s instructions. Phenotypic resistance for first and second line drugs was performed using the MGIT™ 960 System as previously described. Critical concentrations for ofloxacin and kanamycin (second line drugs) were 2.0 pg/mL and 5.0 pg/mL, respectively. Resistance to first and second line drugs was determined using standard diagnostics algorithms. 20 DNA preparation. MTB isolates were handled in blinded fashion throughout. MTB samples (0.5 mL) were pipetted into cryovial tubes containing 1.5 mL PrimeStore Molecular Transport Medium® (a molecular transport medium) (Longhorn Vaccines & Diagnostics, San Antonio, TX). Inactivated samples were transported from South Africa to San Antonio, Texas, USA at ambient temperature (3-4 days) and stored at 25 5°C until used. Total DNA (50 pi) was purified from a 200 pi aliquot of PrimeStore MTM® containing inactivated culture using a Qiagen® EZl® Advanced Robot and EZl DNA Tissue Kit (Cat No. 953034) according to manufacturer’s recommendations (Qiagen Inc., Germantown, MD).
Primer Design. Novel PCR primers were designed for amplification of full-length 30 coding regions for each MTB gene of interest (Table 6).
Table 6 26 ο ο (Ν (Ν
PCR amplification primers used for full length analysis of MTB Genes. Amplification Forward Reverse Target rpoB (Ν mο (Ν
Amplicon (t>p) 1625 2056 2447 960 2664 1680 5' - TCCTCTAAGGGCTCTCGTT -3' 19nt (SEQ ID NO 1) 5’ - GTCAGGTACACGATCTCGT -3' 19nt (SEQ ID NO 2) rpoBll (2 hall·) 5' - ATCGAAACGCCGTACCGCAA -3’ 20nt (SEQ ID NO 3) 5' - TGACGTCGAGCACGTAi\CTCCCT -3' 23nt (SEQ ID NO 4) kalG 5' - ACACCAACTCCTGGGAAGGAAT - 3’ 22nt (SEQ ID NO 5) 10 5' - TGATCGCACATCCAGCACATTT -3' 22nt (SEQ ID NO 6) pncA 5' - GACGGATTTGTCGCTCACTAC -3' 21nt (SEQ ID NO 7) 5' - GCCGGAGACGATATCCAGAT -3' 20nt (SEQ ID NO 8) gyrA 5' - AAGGATGTTCGGTTCCTGGAT -3' 21nt (SEQ ID NO 9) 5' - TAACACTCGTACCCGGCT -3' 18nt (SEQ ID NO 10) 15 ITS (16s) 5' -TTCTAAATACCTTTGGCTCCCT -3' 22nt (SEQ ID NO 11) 5' -TGGCCAACTTTGTTGTCATGCA -3' 22nl (SEQ ID NO 12) (5 genes) 11,432 BP total Primer pairs for rpoB (2 sets of primers), katG, pncA, gyrA, and rrs (16s) gene 20 amplification were designed using the genome sequence of M. tuberculosis H37Rv strain as reference (GenBank accession no. NC_000962). Primer secondary structure, melting temperature, and potential primer-dimer formation were determined using LaserGene 9.1 (DNAStar, Madison, WI) and PrimerExpress 3.0 (Life Technologies, Foster City, CA). All oligonucleotides were synthesized using standard, de-salted 25 primers (Integrated DNA Technologies (IDT), San Diego, CA). PCR Amplification. Amplification reactions for all MTB gene targets were designed and optimized to be used under one standardized set of thermocycling parameters. All PCR ‘mastermixes’ were prepared using Platinum Taq DNA Polymerase, lOX Buffer, and 50 mM MgCh (P/N 10966-034; Life Technologies, Foster City, CA). 30 Amphfication was carried out in a 50 μ1 final volume reaction mixture containing 24.1 μ1 Ambion Nuclease-Free Water (Cat No. AM 9932; Life Technologies, Foster City, CA), 5 μ1 lOX PCR Buffer, 2 μ1 50 mM MgCh (2 mM final), 0.4 μ1 PCR Nucleotide Mix Ultrapure dNTPS (200 μΜ final for each dNTP; P/N 77119; USB, Santa Clara, CA), 0.5 μ1 Platinum Taq DNA Polymerase (2.5 Units final), and 2 μ1 primer blend 35 (rpoB, katG, pncA, gyrA, or rrs genes; 0.4 μΜ final for each primer). To each 34 μ1 ‘mastermix’ reaction mixture, 16 μ1 extracted DNA was added to bring the total volume to 50 μ1. Reactions were carried out in MicroAmp Optical 96-Well Reaction Plates (P/N N801-0560, Life Technologies, Foster City, CA) and capped using MicroAmp 8-Cap Strips (P/N 4323032, Life Technologies, Foster City, CA). 27 r- ο (N δ' Ό (Ν (Ν m ο (Ν
Amplification was performed using an ABI 2720 thermocycler (Life Technologies, Foster City, CA). Thermocycling parameters were 95°C for 2 minutes, followed by 40 cycles at 95°C for 30 seconds, 55°C for 15 seconds, and 72°C for 2 minutes with final extension at 72°C for 5 minutes. Resulting amplicons were confirmed by addition of 5 5 μ1 PCR product with 1 μ1 GelPilot Loading Dye 5X (P/N 1037649; Qiagen, Germantown, MD) on 1% (wt/vol) Molecular Biology Grade Agarose (Cat No. BP1356; Fischer Scientific, Pittsburg, PA) with ethidium bromide (0.1 μg/mL final; Cat No 161-0433; Bio-Rad, Hercules, CA). Electrophoretic separation of products was carried out for 60 minutes at 0.4 mV cm“ in IX Tris Borate-EDTA (TBE) Buffer (Cat 10 No. 1B70153; IBI Scientific, Peosta, lA). Amplicons were visualized under UV transiliumination, and size estimation made using a Trackit 1 kb Plus DNA Ladder (P/N 10488-085; Life Technologies, Foster City, CA). After visualization, the remaining PCR reaction for each clinical isolate gene amplification (~45pL) corresponding to rpoB, katG, pncA, gyrA, and rrs (16s) targets were transferred to a 15 single microcentrifuge tube. Pooled genes corresponding to each clinical isolate were subjected to PCR purification and eluted in 50 μ1 Low Tris-EDTA (TE) (Cat No. 602-1155-010; Life Technologies, Foster City, CA) using the MinElute Reaction Cleanup Kit (Cat No. 28204; Qiagen, Germantown, MD) according to manufacturer’s instructions. The concentration and purity of DNA was determined 20 spectrophotometrically using a NanoDrop ND 1000 (Thenno Fischer Scientific, Wilmington, DE).
Ion Torrent Library Preparation. Barcoded libraries were generated using the Ion Xpress Plus Fragment Library Kit (Cat No. 4471269, Life Technologies, Foster City, CA) and the Ion Xpress DNA Barcoding 1-16 Kit (Cat No. 4468654, Life 25 Technologies, Foster City, CA) according to a modified version of the protocol outlined in the Ion Xpress Plus gDNA and Amplicon Library Preparation.
Amplicon Shearing. Chemical shearing was performed using 1-3 μg DNA containing an approximate equimolar pool of rpoB, katG, pncA, gyrA, and rrs (16s) gene amplicons. DNA shearing was performed in a 50 μ1 total reaction volume by 30 combining 5 μ1 Ion Shear Plus lOX Reaction Buffer, 10 μ1 enzyme, and 35 μ1 pooled DNA template (Ion Xpress Plus Fragment Library Kit, Cat No. 4471269, Life 28 ο (N S'
(N
(N m o (N
Technologies, Foster City, CA). The reaction mixture was incubated at 37°C for 45 minutes, terminated using 5 μ1 Ion Shear Stop Buffer, and stored on ice until purification. Sheared DNA was purified using Agencourt Ampure XP-PCR Purification beads (P/N A63880; Beckman Coulter, Brea, CA) with Dynal magnetic 5 bead stand (Cat No. 123-2ID; Life Technologies, Foster City, CA) according to manufacturer’s recommendations. Briefly, 99 pi Agencourt beads was mixed with 50 pi ion shear reaction, incubated for 5 minutes at room temperature, placed on a magnetic stand, washed twice with 70% (v/v) ethanol, and eluted using 12 pi Low TE Buffer (Cat No. 602-1155-010; Life Technologies Inc., Foster City, CA). 10 Adaptor Ligation. Adaptor ligation was performed in a 0.2 mL low bind PCR tube (P/N PCR-02-L-C; Axygen Inc., Union City, CA) by combining 12 pi sheared amplicon with 1.25 pi Ligase Buffer, 1.25 pi Pl-IA Adaptor Mix (Ion DNA Barcoding 1-16 Kit, Cat No. 4468654 Life Technologies, Foster City, CA) and 0.2 pi DNA Ligase (Ion Xpress Plus Fragment Library Kit, Cat No. 4471269, Life Technologies, 15 Foster City, CA). The mixture was pipetted up and down 5 times and incubated at room temperature (22-25°C) for 30 minutes. Adaptor ligation reactions were purified and eluted in 10 pi Low TE Buffer using the Agencourt Ampure XP-PCR Purification beads (P/N A63880; Beckman Coulter, Brea, CA) with the Dynal magnetic bead stand (Cat No. 123-21D; Life Technologies, Foster City, CA) according to manufacturer’s 20 recommendations..
Nick Translation and Barcode Amplification. Amplicon pools from each patient sample were barcoded using the Ion DNA Barcoding 1-16 Kit and Ion Xpress Fragment Library Kit (Part Nos. 4468654 and 4471269, respectively; Life Technologies, Foster City, CA). To maximize yields reactions were scaled 2x by 25 combining 40 pi Platinum PCR SuperMix High Fidelity, 4.4 pi of Ion Primer Mix (BC X where X= barcode 1-16) and 10 pi of ligated DNA. Amplification was performed using an ABI 2720 thermocycler (Life Technologies, Foster City, CA). Thermocyciing parameters comprised 72°C for 20 minutes, 95°C for 5 minutes, followed by 10 cycles of 95°C for 15 seconds, 58°C for 15 seconds and 68°C for 1 minute. Following 30 amplification, bar-coded samples were purified and eluted in 50 pi of Low TE (Cat No. 602-1155-010; Life Technologies, Foster City, CA) using the MinElute Reaction 29
ο (N
(N
(N m o (N
Cleanup Kit (Cat No. 28204; Qiagen, Germantown, MD) according to manufacturer’s instructions. DNA concentration and purity was determined by spectrophotometi'ic analysis using a NanoDrop ND 1000 (Thermo Fischer Scientific, Wilmington, DE). Ranges for purified bar-coded samples are typically 150-300 ng/μΐ with A260/280 5 purity of 1.7-1.9. Equimolar concentrations (~2-3 μg of each bar-coded sample) were combined into a single 1.5 mL nuclease-free microcentrifuge tube and used for size selection.
Size Selection. The appropriate volume of GeiPilot 5X Loading Dye (P/N 1037649; Qiagen, Germantown, MD) was added to the pooled bar-coded MTB library tube and 10 loaded onto a 1 % (w/v) agarose gel (Cat No. BP1356; Fischer Scientific, Pittsburg, PA) containing ethidium bromide (0.1 pg/mL final; Cat No 161-0433; Bio-Rad, Hercules, CA). The bar-coded library was electrophoresed for 60 minutes at 0.4 mV cm^ in IX TBE Buffer (Cat No. 1B70153; IBI Scientific, Peosta, lA) and visualized under UV transillumination. Size estimations were determined using a Trackit 1 kb 15 Plus DNA Ladder (P/N 10488-085; Life Technologies, Foster City, CA). Gel excision was performed under UV transillumination using a sterile scalpel blade excising out a target region between 75-200 bp. Excised agarose gel slices were placed into sterile 1.5 mL microcentrifuge tubes and subjected to DNA purification using the PureLink Quick Gel Extraction Kit (Cat No. K210012; Life Technologies, Foster City, CA) 20 according to manufacturer’s instructions. Concentration and purity values for the barcoded DNA library were determined spectrophotometrically using a NanoDrop ND 1000 (Thermo Fischer Scientific, Wilmington, DE). The recommended library input for emulsion PCR is -140-560 x 10® molecules per 18 μ1. This range was achieved by a 1:1000 dilution using library stock and nuclease-free water.
25 Emulsion Polymerase Chain Reaction (emPCR). Emulsion Polymerase chain reaction was performed in a 1 mL reaction volume using the Ion Template Preparation Kit (Cat No. 4469000; Life Technologies, Foster City, CA) by adding 582 μ1 nuclease-free water, 200 μ1 5X PCR Reagent Mix, 100 μ1 lOX PCR Enzyme Mix, 100 μ1 Ion Sphere Particles, and 18 μ1 diluted library template. The preparation w'as mixed thoroughly 30 followed by brief centrifugation in a microcentrifuge. Emulsion was achieved using the Ultra-Turrax Tube Drive (Life Technologies, Foster City, CA). A total of 9 mL 30
ο (N
(N
(N m o (N chilled Emulsion Oil (Ion Torrent Preparation Kit; Cat No. 4469000, Life Technologies, Foster City, CA) was added to an Ion Template Preparation Tube (Cat No. 4467226, Life Technologies, Foster City, CA). The emulsion tube was placed and locked onto the IKA Ultra-Turrax Tube Drive and initiated. While the tube was in 5 motion, the entire 1 mL PCR master mix solution was dispensed into the cap port and mixed for 5 minutes. The mixed emulsion was transferred to a 96-well PCR plate and amplified using an ABI 2720 thermocycler (Life Technologies, Foster City, CA) using the following thermocycling parameters: 94°C for 6 minutes, followed by 40 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 90 seconds; followed by 5 10 cycles at 94°C for 30 seconds, and 68°C for 6 minutes.
Ion Sphere Particle (ISP) Recovery and Qubit Measurement. Ion Sphere Particles were recovered using reagents supplied in the Ion Xpress Template Kit (Cat No. 4469001, Life Technologies, Foster City, CA) according to manufacturer’s protocol (Ion Xpress Template Kit User Guide v2.0, pages 18-19). Quantification of recovered 15 particles was performed using a Qubit 2.0 Fluorometer (Life Technologies, Foster City, CA) and an Ion Sphere Quality Control Kit (Cat No. 4468656, Life Technologies, Foster City, CA) according to manufacturer’s recommendations (Ion Xpress Template Kit User Guide, page 25-26). The optimal amount of template-positive ion sphere particles (ISPs) is between 4-50%. Relative fluorescent unit (RFU) values obtained 20 outside of this range were not pursued into subsequent ISP enrichment. ISP Enrichment. ISPs were enriched using reagents supplied in the Ion Xpress Template Kit, Ion Sequencing Kit, and DynaBeads MyOne Streptavidin Cl beads (Cat Nos. 4469001, 4468997 and 650.01 respectively; Life Technologies, Foster City, CA) according to the manufacturer’s protocols (Ion Xpress Template Kit User Guide v2.0, 25 pages 15-17).
Ion Torrent 314 Chip Preparation and PGM Sequencing. Ion Torrent 314 Chips (Cat No. 4462923; Life Technologies, Foster City, CA) were prepared and loaded according to manufacturer’s recommendation (Ion Sequencing Kit User Guide v 2.0). The Ion Torrent PGM was run according to Ion Torrent 314 Chip specifications 30 including a 65-cycle sequencing protocol, use of 18 megaOhm purified water, and standard compressed argon gas to drive fluidics through the PGM system. All rpoB, 31 ο (N S'
(N
(N H o (N katG, pncA, gyrA and rrs genes and corresponding proteins were deposited into GenBank (accession numbers JX303203-JX303332).
Gyrase PCR for the Detection of TB vs. 6110 PCR assay. The gyrase target for OCR and whole Gyrase gene sequencing on the Ion Torrent PGM can also be used to 5 identify TB mutations that lead to resistance. This second PCR target allows for the accurate analysis of TB strains that may not include the entire IS6110 insertion element. While the IS6110 assay has multiple gene copies in most strains, some have only one. As shown in Figures 6, 7 and 8, this Gyrase assay has a generally higher cycle threshold in comparison to the IS6110 assay due to multiple IS6110 gene copies 10 in those isolates and thus more sensitivity. Thus any possible TB mutation can be followed- even away from the detection site by this method of full gene sequencing. Phenotypic and genotypic results. Amino Acid characterization of 26 M. tuberculosis isolates by Ion Torrent sequencing of rpoB, katG, pncA, gyrA, and rrs (16s) genes are summarized in Tables 1-5, respectively, and compared to BACTEC™ 15 MGIT™ 960 (phenotypic), and/or HAIN GenoType® MTBDRplus (genotypic) LPA. Of the 26 MTB clinical isolates, 14 (54%) were MDR, 7 (21%) were XDR, and 5 (19%) were sensitive to drugs by BACTEC^”^ MGIT™ 960 phenotypic analysis. The Ion Torrent PGM sequencing method showed 100% (26/26) concordance to both phenotypic resistance obtained by MGIT'^^‘ 960 culture (Tables 1-5) and genotypic 20 rpoB and katG data obtained by Main LPA (Table 1, 2). rpoB gene mutations. A total of 10 rpoB amino acid substitutions were identified in the 26 clinical isolates compared to the H37Rv wild type strain. The common S531L mutation was the most prevalent, but mutations in codons 516 and 526, also known to confer resistance to rifampin w'ere observed (Table 1). Additionally, mutations were 25 observed within the rpoB open reading frame but outside of the 81-basepair rifampin resistance-determining region (RRDR; Table 1). The V194I mutation observed outside of the RRDR in one strain is a unique substitution that is likely not associated with rifampin resistance. Five amino acid substitutions were noted in at least one strain beyond residue position 900 of the rpoB protein. There were seven strains with an 30 rpoB mutation (6 at position 516 and 1 at position 526) where a wild type band was absent without a corresponding mutation band according to LPA. In six of these seven 32
ο (N
(N
(N H o (N isolates, Ion Torrent sequencing revealed an uncommon amino acid substitution (i.e., glycine) within a known mutation site at position 516 where a valine (V) substitution (D516V) is typically known to occur (Table 2). Similarly, in one isolate Ion Torrent sequencing revealed an arginine (R) within a known mutation site at position 526 5 where tyrosine (Y) or aspartic acid (D) substitutions (H526Y/D) typically occur. katG gene mutations. Four amino acid substitutions were observed in the katG gene with S315T which is known to confer isoniazid resistance present in all resistant strains (Table 2). Clinical strains harboring R463L, W191R, and N138H mutations were detected by DST (Table 2) and have been previously characterized. A substitution at 10 position 463 (R463L) in katG has been previously shown to have no effect on antibiotic resistance and can be used to categorize M. tuberculosis isolates into genetic Groups 1 (Arg463) or 2/3 (Leu463). Of 26 clinical isolates assessed, 7 (27%) were members of genetic Group 1 as evident by this R463L substitution. pncA gene mutations. Seven nucleotide mutations were noted in at least one strain 15 among 561 bps comprising the full-length coding region for the pncA gene (Table 3). Nine of 26 strains (34.6%) contained an amino acid mutation conferring pyrazinamide resistance (Table 3). In one strain, a silent (synonymous) nucleotide mutation was characterized at nucleotide position 195 (C195T). Five strains contained previously characterized amino acid substitutions (C14R, A102V, V139G, R154G, and L172P) 20 known to confer resistance to pyrazinamide. A novel mutation, not previously reported elsewhere, encoding a termination stop codon was found in one isolate at residue 122 (Q122Stop) in the pncA protein (Table 3). gyrA gene mutations. Nine unique mutations were observed in the 2,517 bp full-length gyrA gene encoding subunit A of the DNA gyrase enzyme. Resistance to 25 fluoroquinolones (FQ) was only noted in strains harboring mutations in the quinolone resistance determining region (QRDR) defined by substitutions in gyrA at codons 88, 90, and 94. A number of additional mutations were also observed in regions outside of the QRDR including two ‘mixed strain’ mutations at position 549 and 613 in the gyrA protein (Table 4). Mutation at position 95 (S95T) is known to have no effect on FQ 30 resistance but can be used to categorize strains in genetic Groups 2 or 3. Of the 19 total clinical isolates belonging to genetic Groups 2/3, 18 (96%) were Group 2, and 1 (4%) 33 ο (N S'
(N
(N m o (N was Group 3 according to assessment of gyrA position 95 (T = genetic Group 2, and S = genetic Group 3). rrs (16s) gene mutations. Four nucleotide mutations were noted among the 1,540 bps comprising the full length 16s rrs gene. Seven of 26 (27%) clinical isolates were 5 shown to be resistant to aminoglycosides by DST, and all strains harbored an A1401G mutation known to confer resistance (Table 5). Two other amino acid mutations (C492T and A514C) were observed, but have been previously shown to not inhibit aminoglycoside efficacy. A previously uncharacterized G878A nucleotide mutation was observed, but the isolate was shown to be sensitive according to DST (Table 5). 10 Megabase Sequencing. Ion torrent gene chip sequencing was performed on the complete genome of Influenza virus A under five distinct conditions, identified in Figure 9 as Tracks. Whole viral nucleic acid of Influenza A, strain H3N2 (about 14.4 kb total RNA) was prepared as discussed above and either reverse transcribed only, or reverse transcribed and PCR amplified as indicated in Figure 9. Influenza virus 15 genome was mass amplified by reverse transcription (RT) and certain amplified cDNA populations subjected to PCR. Each result was then analyzed using the Ion Torrent sequencing protocol. RT and/or RT-PCR analysis was performed with uniform hexamers, Uni 12, and/or 24 different influenza-specific primers (different in both length and sequence). Uniform hexamers comprise a collection of primers, each six 20 nucleotides in length whereby the collections contain ail of sequence iterations of the six nucleotides. Uni 12 is primer that contains a sequence that is complimentary to 12 nucleotides at the 3' terminus of each of the segments of the influenza H3N2 viral genome (5'~ACGCGTGATCAGCAAAAGCAGG; SEQ ID NO 13). As shown in Figure 9, Track 4 amplification and sequencing with hexamer primers and Uni 12 25 followed by PCR amplification with the 24 influenza-specific primers and Ion Torrent protocol sequencing identified about 70% of the influenza genome.
Additional experiments were performed to achieve one-step sequencing of the complete Influenza genome. A series of influenza-specific primers were developed that would allow for uniform conditions to be performed for a PCR reaction. The primers 30 that were developed are listed in Figure 10. These primers are all specific for the influenza virus genome with primer pairs spaced along the genome about every 800 to 34 r-
ο (N
Ό (N
(N mo (N 10 15 20 1,000 bases in length (see Figure 10, ampiicon length and start and stop positions for primer placement and sequence). All primers were of similar length, about 18-23 nucleotides and contain a similar GC content, about 22.7% to 38.9%, with nearly about 33% ±6% and most about 33% +3%. PCR analysis was performed using different collections of these primers and the amplified products identified using the Ion Torrent sequencing protocol. Sequencing of pncA gene. The gene sequence of pncA for PZA resistance was determined using a series of primers spaced or “tiled” along the pncA gene in accordance with the disclosure and compared to results achieved with traditional Sanger sequencing. The coding sequence of the pncA gene is depicted in Figure llA and the primers utilized are depicted in Figure 1 IB in bold and underlined. Using these primers in conjunction with Ion Torrent methodology, the entire coding regions of pncA was determined (see P1-P4 of Figure IIB). Expanding the primers utilized to all genes or of specific regions allows for one-step sequencing of the entire genome. The surprising results achieved identified 2 or 11 cases of mixed strain (heterogenous) populations that contain both wild-type and mutant that would have been missed by traditional Sanger sequencing. A summary of the amino acid mutations in pncA of MTB clinical isolates deduced by Ion Torrent according to the methods of the disclosure is shown in Table 7 and can be compared with Table 8 showing the results achieved with Sanger sequencing.Table 7
Samnle No. oncA Mutation (561 bp)=^* Phenotvne NT3346 INDEL deletion of T at pos 12 causes stop at pos 4 Resistant NT661/1 Missense INDEL deletion of T at pos 582 Resistant 25 ML1632/2 promoter, insert T after C at pos 12 Resistant Sz-426/12 wildtype Sensitive FS4751103/1 Missense INDEL G inserted at pos 35 Resistant W3797/2 D12G* Resistant S2744 H51D Resistant 30 ML1440/2 S59P* Resistant EC2248/1 A79V* INDEL at Stop 127, insert at pos 360 in 82% of strains Resistant ML2482/1 K96STOP* Resistant 35 WC2601/2 T135P* (seen in 61%) and silent T to C at pos 475 in 25% Resistant 35
r-ο (N * - There is a known heterogenous nucleotide mutation that confers mixed amino acid Ό (N (N H O (N substitution ** = In comparison to H37Rv reference strain 5 Table 8 Sample No. uncA Mutation (561 bp)** Phenotvue NT3346 Insertion 12, STOP55 Resistant NT661/1 Missense INDEL deletion of T at pos 582 Resistant ML1632/2 promoter, insert T after C at pos 12 Resistant 10 Sz-426/12 ??? ??? FS4751103/1 Missense INDEL G inserted at pos 35 Resistant W3797/2 D12G* Resistant S2744 ??? Resistant ML1440/2 Wildtype Resistant 15 EC2248/1 A79V and Stop 126 Resistant ML2482/1 K96STOP Resistant WC2601/2 wildtype Resistant ** = In comparison to H37Rv reference strain 20
As shown in the comparison of Table 7 with Table 8, WC2601/2 showed a T135 mutation had no corresponding mutation by Sanger sequencing. The mutation was heterogeneous with 61% of cells containing the mutation with 29% as wildtype. With ML1440/2, a S59P mutation was identified with no corresponding mutation by 25 Sanger sequencing. The mutation was heterogeneous with 95% containing the mutation with 5% wild-type.
Rapid characterization of drug resistance genes directly from patient sputum samples. The methods of the disclosure address a need for performing rapid characterization of drug resistance genes from patient sputum samples obtained from, 30 for example, remote areas. The method includes collection to analysis of MTB rpoB and pncA genes that confer resistance to first line drugs, rifampicin and pyrazinamide, respectively. The methodology employs ambient temperature shipment of sputum in PrimeStore Molecular Transport Medium (MTM), nucleic acid extraction, gene amplification and sequencing directly from sputum for MTB drug resistance 35 characterization.
Sputum specimens were collected as part of a large prospective analysis of MTB diagnosis in rural South Africa (patients in Mopani, South Africa). For molecular 36
ο (N
Ό (N
(N m o (N r- testing, a flocked swab (Copan Diagnostics, Brescia, Italy) was submerged and swirled a minimum of five times in sputum and then subsequently transferred into 1.5 mL of molecular transport medium, PrimeStore MTM® (PS-MTM). PS-MTM is a clinical transport solution that inactivates microbes including M. tuberculosis, and preserves 5 and stabilizes released RNA/DNA for safe, ambient temperature shipment. PS-MTM tubes containing sputum were all shipped from South Africa to a fully equipped facility in San Antonio, Texas at ambient temperature using a commercial carrier.
Total genomic DNA was purified using the PrimeXtract kit (Longhorn Vaccines and Diagnostics, San Antonio, TX, USA) according to manufacturer’s 10 recommendations. Real-time PCR amplification of MTB was performed using PrimeMix TB® (PM PCR), an all-inclusive reagent blend that targets the highly conserved MTB IS6110 region. PCR amplification using MTB primers for pncA and rpoB were performed as previously described. Primers for rpoB (1,625 bps) and pncA (960 bps) amplify a 15 portion of the gene containing the full rpoB determining region and the promoter plus full coding region of the pncA gene, respectively. For NGS library preparation, pncA and rpoB gene amplicons were prepared using the Nextera XT Sample Prep Kit. MiSeq NGS was performed according to manufacturer’s instructions (Illumina, San Diego, CA, USA) using MiSeq Reagent Kit (V3) with 600 cycles. Bioinformatics were 20 performed using SeqMan NGen (V8) and LaserGene (V12) Core Suite (DNAStar, Inc, USA) with genetic comparison to the H37Rv reference strain.
Of the 22 specimens selected for rpoB and pncA NGS, 17 {113%) produced complete DNA sequence (Table 9). A total of five samples were omitted due to partial gene sequencing, poor sequence quality, or low coverage depth (i.e., below lOX). 25 Specimens producing full sequence had PCR real-time values ranging from 23.5 to 37.4, with the majority having CT values less than 30. Success in obtaining quality NGS from original specimens hinges on the concentration of MTB recovered during extraction. Using a qualitative real-time PCR assay prior performing endpoint amplification of MTB resistance genes may be predictive of NGS success. In three 30 specimens NGS do not produce suitable data, most likely due to inefficient amplification in the longer 1625 bp rpoB PCR amplicon (Table 9). 37
ο (N
Table 9 Ion Torrent Sequencing* of MTB rpoB and pncA gene from selected patient sputum testing positive by Primemix MTB real-time PCR (N-22) Ό (N (N cn o (N Primemix ion torrent sea’in 2 mutations 5 Patient RT-PCR Xnert/RIF MGIT rpoB pncA 104 -t- (CT=23.5) + -F wt wt 64 + (CT=25.2) -F -F wt wt 117 -f (CT=25.4) -f/RIF** res -F H-526-D# wt 54 + (CT=26.0) -F - C-309-T# wt 10 47 -1- (CT=26.7) + -F wt wt 119 -t (CT=27.2) + + wt wt 83 -t- (CT=28.3) -F -F wt wt 74 + (CT=28.8) + + wt wt 71 -t- (CT=28.9) -f/RIF** res -F H-526-Y# wt 15 89 + (CT=28.9) -F -F V-194-1# wt 81 -f (CT-29.5) -F -F wt wt 50 + (CT=30.7) -f/RIF** res + H-526-Y# wt 85 -F (CT=33.8) -F c NA NA 72 -t- (CT=34) + - NA wt 20 134 -F (CT=34.4) + -F NA NA 2 -F (CT=35.2) + - NA wt 127 -F (CT=36.0) + + NA NA 20 -F (CT=36.0) + c wt wt 10 -F (CT=37.4) + - wt wt 25 no + (CT=38.0) + - NA NA 108 -F (CT=38.7) + - NA R-2-P## 120 -F (CT=39.4) + - NA NA * = lUumina MiSeq to amplify the full 561 base pair pncA gene plus 45 base pairs promoter region 30 base pairs) and a 1608 bp rpoB gene region that included the rpoB determining region. ** RIF = rifampin resistant Wt = wild-type according to H37Rv strain. NA = sequence no available # mutation at position 526 of the rpoB gene is known for resistance mutation . 35 ## mutation at position 2 of the pncA gene (iU‘ginine-2-piOline).
Resistance mutations were found in rpoB gene sequences which correlated with those determined by Xpert. Upon rpoB gene NGS characterization, three specimens contained classical resistance mutations at position 526 of the rpoB determining region. 40 Interestingly, two specimens contained a H-526-Y and one a H-526-D mutation (Table 9). A V-194-1 substitution was observed in one specimen (Patient 89) that has been shown previously to be a non-resistance conferring mutation. A synonymous silent mutation, i.e., C-309-T was noted in the rpoB of one specimen. The pncA gene sequences from all stiains were found to be wild type in comparison to the H37RV 38
ο (N
(N
(N m o (N r- reference strain (Table 9), with the exception of a novel R-2-P mutation in one specimen. It is not known whether this mutation confers resistance to pyrazinamide, and since it was not detected by Xpert or MGIT culture no drug resistance data is available for this specimen. The patient from whom this specimen was derived 5 presented with persistent cough and weight loss. Follow up testing of this patient using real-time PCR was low positive (CT= 36.1) but Xpert and MGIT culture were negative.
The ability to improve MTB detection with sensitive real-time PCR and then rapidly sequence resistance genes provides another opportunity for low resource areas. Since PS-MTM rapidly kills MTB and preserves the DNA at ambient temperature and 10 above, specimens can be efficiently transported, for real-time PCR and sequencing to improve detection of drug resistant strains and optimize patient therapy. Previous studies have shown the benefit of sequencing MDR strains from patients who have come to the US from countries with MDR and XDR to identify known and new resistance mutations. An additional advantage of NGS is that the depth of coverage 15 provides the ability to detect more tlian one population, i.e., heteroresistance in the patient's specimen. Heteroresistant characterization is important for patient care, especially if MTB subpopulations that are resistant to key antibiotics as these become the predominant patient strain. This example also demonstrates the feasibility of transporting sputum specimens efficiently to central and regional labs to provide 20 support to rural clinics. Without adding extra training staff or infrastructure, patient sputum specimens from rural areas can be transported to labs with highly trained personnel and state of the art equipment to support MTB patient care surveillance and research.
Characterization of Mycobacterium tuberculosis (MTB) drug resistance genes is 25 critical for the appropriate treatment of tuberculosis (TB). Molecular detection and next-generation sequencing (NGS) are rapidly providing new tools to diagnose and improve treatment of drug resistant TB. Understanding the epidemiology and the role of mobile populations in rapidly changing resistance patterns, particularly in rural African settings is important as we work to treat and eradicate TB. In this brief report, 30 NGS was used to characterize MTB rpoB and pncA drug resistance genes directly from sputum collected and transported at ambient temperature from mral South Africa to 39
r-ο (N
(N
(N mo (N 10
Texas in PrimeStore® MTM (PS-MTM). These genes confer resistance to first line drugs, rifampicin and pyrazinamide, respectively. This work is significant because stable specimens containing high quality DNA enable rapid, centralized processing directly from sputum. Other embodiments and uses of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the disclosure indicated by the following claims. 40

Claims (37)

  1. CLAIMS;
    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 with multiple primer pairs, wherein each primer pair of the multiple primer pairs comprise primers that are from 15 to 25 nucleic acids in length, have a GC content of about 25%-50%, and an annealing temperature within 5°C; obtaining sequence information of the amplified sequences by next generation sequencing, wherein the amplified sequences represent overlapping portions of the complete sequence of the organism; 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.
  2. 2. The method of claim 1, wherein the motif is indicative of the presence of a pathogen.
  3. 3. The method of claim 2, wherein the organism is one or more of a virus, a bacterium, a fungus or a parasite.
  4. 4. The method of claim 3, wherein the virus is one or more of a DNA virus, an RNA virus, a positive or negative single-strand virus, a double strand virus, an orthomyxovirus, a paramyxovirus, a retrovirus, a flavivirus, a filovirus, a lentivirus, an influenza virus, a human immunodeficiency virus, a hepatitis virus, or an ebola virus.
  5. 5. The method of claim 3, wherein the bacterium is Mycobacterium tuberculosis, Plasmodium falciparum, FranciseUa tularensis, Yersinia pestis, or Vibrio cholera.
  6. 6. The method of any one of claims 1 to 5, wherein the biological sample is bodily fluid and/or tissue obtained from a patient.
  7. 7. The method of any one of claims 1 to 6, wherein the motif does not specifically hybridize to other nucleic acid sequences of the organism.
  8. 8. The method of any one of claims 1 to 7, where the samples are provided in a molecular transport medium and the molecular transport medium contains a chaotrope, a detergent, a reducing agent, a chelator, a buffer, and an alcohol, together present in an amount sufficient to lyse cells, denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid.
  9. 9. The method of any one of claims 1 to 8, wherein samples are provided at ambient temperatures without refrigeration.
  10. 10. The method of any one of claims 1 to 9, wherein the next generation sequencing is ion torrent sequencing.
  11. 11. The method of claim 10, wherein the ion torrent sequencing is performed in a single reaction.
  12. 12. The method of any one of claims 1 to 11, further comprising at least two motifs, wherein the presence or absence of the two amplified motifs is determined together.
  13. 13. The method of any one of claims 1 to 12, wherein the motif contains a region that encodes an antimicrobial gene sequence.
  14. 14. The method of claim 13, wherein the antimicrobial gene sequence encodes an antibiotic.
  15. 15. The method of any one of claims 1 to 14, wherein the polymerase chain reaction is carried out in an aqueous mix comprising: a polymerase and optionally a reverse transcriptase; a mix of deoxynucleotide tri phosphates comprising about equivalent amounts of dATP, dCTP, dGTP and dTTP, a chelating agent, an osmolarity agent, an albumin, a magnesium salt; and a buffer.
  16. 16. A method for rapidly determining a complete sequence of a target gene or genome comprising: 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 is from 15 to 25 nucleic acids in length, has a GC content of about 25%-50%, and has a similar annealing temperature; sequencing each of the series of amplicons produced by next generation sequencing in a single reaction wherein the amplicons represent overlapping portions of the complete sequence of the target gene or genome, and correlating the sequences of the amplicons and constructing the complete sequence of the target gene or genome.
  17. 17. The method of claim 16, wherein the target gene or genome is RNA and the target is reverse transcribed to DNA before performing PCR.
  18. 18. The method of claim 16 or claim 17, wherein each of the primers of the multiple primer pairs comprise primers that are from 15 to 25 nucleic acids in length and has a GC content of about 25%-50%.
  19. 19. The method of any one of claims 16 to 18, wherein each primer pair is designed to PCR amplify an amplicon, and the collection of amplicons that are PCR amplified encompass overlapping segments of the complete sequence of the target.
  20. 20. The method of any one of claims 16 to 19, wherein the plurality of primer pairs hybridizes to the target with primer pairs spaced along the target at about every 500 to 2,000 nucleotides.
  21. 21. The method of any one of claims 16 to 20, wherein the target gene or genome is of an organism and the organism is a virus, a bacterium, a fungus, a parasite or a cell.
  22. 22. The method of claim 21, wherein the virus is one or more of a DNA virus, an RNA virus, a positive or negative single-strand virus, a double strand virus, an orthomyxovirus, a paramyxovirus, a retrovirus, a flavivirus, a filovirus, a lentivirus, an influenza virus, a human immunodeficiency virus, a hepatitis virus, or an ebola virus.
  23. 23. The method of claim 21, wherein the bacterium is one or more of Mycobacterium tuberculosis, Plasmodium falciparum, Francisella tularensis, Yersinia pestis, or Vibrio cholera.
  24. 24. A method for determining the sequence of a nucleic acid target in one cycle of steps comprising: 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 is from 15 to 25 nucleic acids in length, has a GC content of about 25%-50%, and 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.
  25. 25. The method of claim 24, wherein the nucleic acid of the sample is RNA and the RNA is reverse transcribed prior to PCR.
  26. 26. The method of claim 24 or claim 25, wherein the nucleic acid target is greater than 1 Mb in length.
  27. 27. The method of any one of claims 24 to 26, wherein each of the primers of the multiple primer pairs is from 16 to 24 nucleotides in length, has a GC content of about 28-35%, and an annealing temperature of within 3°C of each other primer of the multiple primer pairs.
  28. 28. The method of any one of claims 24 to 27, wherein each primer pair is designed to PCR amplify an amplicon representing a portion of the sequence of the nucleic acid target, and the collection of amplicons that are PCR amplified represent overlapping portions of the complete sequence of the target.
  29. 29. The method of any one of claims 24 to 28, wherein the plurality of primer pairs hybridizes to the target at a spacing of about 800 to 1,500 nucleotides in length.
  30. 30. A mixture comprising multiple pairs of nucleic acid primers wherein each primer is from 15 to 25 nucleic acids in length and has a GC content of about 25%-50%, and 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.
  31. 31. The mixture of claim 30, wherein each primer of the collection of primer pairs has a GC content of about 25%-45%, and an annealing temperature to the target within 3°C of each other primer.
  32. 32. The mixture of claim 30, wherein the target is a gene or genome of a microorganism.
  33. 33. The mixture of claim 32, wherein the gene is a gene that confers antibiotic resistance of the microorganism.
  34. 34. The mixture of claim 32, wherein the microorganism is a virus, a bacterium, a parasite, or a fungus.
  35. 35. The mixture of any one of claims 30 to 34, which contains a heat-stable composition comprising”: a polymerase; a mix of deoxynucleotide tri phosphates comprising dATP, dCTP, dGTP and dTTP; a chelating agent; a salt; a buffer; a stabilizing agent and nuclease-free water.
  36. 36. The mixture of any one of claims 30 to 35, wherein the nucleic acid primers do not self-hybridize.
  37. 37. The method of any one of claims 30 to 36, wherein the primer pairs to two or more genes are multiplexed together.
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