WO2021243333A2 - Test d'acide nucléique de masse - Google Patents

Test d'acide nucléique de masse Download PDF

Info

Publication number
WO2021243333A2
WO2021243333A2 PCT/US2021/035240 US2021035240W WO2021243333A2 WO 2021243333 A2 WO2021243333 A2 WO 2021243333A2 US 2021035240 W US2021035240 W US 2021035240W WO 2021243333 A2 WO2021243333 A2 WO 2021243333A2
Authority
WO
WIPO (PCT)
Prior art keywords
cov2
pathogen
oligonucleotide
primer
cdna
Prior art date
Application number
PCT/US2021/035240
Other languages
English (en)
Other versions
WO2021243333A3 (fr
Inventor
David Troy MOORE
Katherine Elena VARLEY
Original Assignee
Biohsv Holdings, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biohsv Holdings, Inc. filed Critical Biohsv Holdings, Inc.
Priority to EP21813023.5A priority Critical patent/EP4157300A2/fr
Publication of WO2021243333A2 publication Critical patent/WO2021243333A2/fr
Publication of WO2021243333A3 publication Critical patent/WO2021243333A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present disclosure relates generally to the detection of specific nucleic acids, and specifically to processes and compositions of matter for use therewith.
  • PCR techniques are reasonably accurate, they are overly specific. PCR diagnostic techniques typically detect one pathogen sequence with very high specificity. This results in false negative results when a pathogen mutates in the target region. Often a mutation of just one base pair in the primer binding region or the probe binding region will result in a negative result. Viruses and microorganisms have high mutation rates, and this problem is especially severe in RNA viruses. In addition, PCR techniques are not easily scalable up to samples pooled from scores or hundreds of subjects.
  • the following discloses a method for effectively screening large pooled samples from many subjects that is sensitive enough to small quantities of genetic material from a pathogenic agent ("pathogen”) that it is potentially able to detect a single pathogen sequence when the genomes of dozens of subjects are present in the pooled sample; and tolerant of unanticipated mutations in the pathogen.
  • pathogen pathogenic agent
  • This is accomplished by using massive parallel sequencing technology (“next gen sequencing”) on samples prepared by patch-ligation techniques. Both techniques were previously used on individual samples, but not to detect potentially small quantities of target pathogen RNA in a sample pooled from many individuals.
  • a method of screening for an RNA pathogen in a population of subjects comprising: creating a cDNA library of a pooled sample from multiple subjects in the population, specifically conjugating sequences from the pathogen with an exonuclease resistant group, digesting the remaining sequences with an exonuclease, specifically amplifying the target sequences, and sequencing the target amplicons by massive parallel sequencing.
  • a method of screening for an RNA in a population of samples comprising: creating a cDNA library from a sample containing RNA, using a 3' adapter oligonucleotide that functions as a reverse transcription primer that will hybridize with a target sequence present in the RNA, wherein the 3' adapter oligonucleotide comprises: a sample barcode identifying the sample, and a first PCR primer binding site; pooling the cDNA library with a plurality of additional cDNA libraries, to create a pooled cDNA library; (c) creating a cDNA digest of the pooled cDNA library, wherein the cDNA digest comprises cDNA fragments; (d) joining a second adaptor oligonucleotide to the cDNA fragments at the 5' end of said cDNA fragments, said second adapter oligonucleotide comprising: a pool barcode identifying the pool, and a second PCR primer binding
  • a method of screening for a pathogen in a population of subjects comprising: creating an amplicon(s) of a pooled sample, specifically conjugating sequences from the pathogen with an exonuclease resistant group, digesting the remaining sequences with an exonuclease, amplifying the target sequences, and sequencing the target amplicon by massive parallel sequencing.
  • a method of screening for a pathogen in a population of subjects comprising: creating a cDNA library by performing RT-PCR on a sample of multiple RNA molecules taken from an individual in the population, using a 3' adapter oligonucleotide that functions as a reverse transcription primer, wherein the 3' adapter oligonucleotide comprises: i. a sequence capable of selectively hybridizing with a target sequence present in the pathogen RNA, ii. a sample barcode identifying the individual, and iii.
  • the cDNA library comprises a plurality of double stranded DNA molecules, each comprising the sample barcode and the first PCR primer binding site; pooling the cDNA library with a plurality of additional cDNA libraries, to create a pooled cDNA library; creating a cDNA digest of the pooled cDNA library; specifically hybridizing a patch oligonucleotide to the cDNA fragments originating from the pathogen at the 5' end of said cDNA fragments; hybridizing a second adaptor oligonucleotide to the patch oligonucleotide while the patch oligonucleotide is hybridized to the cDNA fragments, said second adapter oligonucleotide comprising i.
  • a sequence capable of selectively hybridizing to the patch oligonucleotide ii. a pool barcode identifying the pool, and iii. a second PCR primer binding site; and amplifying the DNA fragments originating from the pathogen using a first primer that hybridizes with the first primer binding site and a second primer that hybridizes with the second primer binding site to create a target amplicon.
  • a method of generating a pathogen-specific amplicon from a population of subjects comprising: creating a DNA digest from a pooled sample of multiple DNA amplification products created from multiple individuals in the population, wherein each of the multiple DNA amplification products comprises a sample barcode identifying the individual and a first PCR primer binding site at a terminal end, and wherein the DNA digest comprises multiple DNA fragments originating from the pathogen; specifically hybridizing a patch oligonucleotide to the DNA fragments originating from the pathogen; hybridizing an adaptor to the patch oligonucleotide while the patch oligonucleotide is hybridized to the DNA fragments, said adapter comprising a second PCR primer binding site; and amplifying the DNA fragments originating from the pathogen using a first primer that hybridizes with the first primer binding site and a second primer that hybridizes with the second primer binding site to create a target amplicon.
  • a method of contagion control comprising screening a population according to any of the first through fifth aspects, detecting at least one sequence from a pathogen in the pooled sample, and implementing a contagion control measure in response.
  • a target amplicon that is a product of the method of any of the first through fifth aspects.
  • a kit for the detection of an RNA in a sample comprising: a reverse transcription primer comprising a sequence capable of selectively hybridizing with a target sequence present in a pathogen RNA, a sample barcode sequence for identifying an individual test subject, and a first PCR primer binding site; a patch oligonucleotide that hybridizes under stringent conditions to a first genomic sequence from the pathogen; and an adaptor oligonucleotide configured to hybridize with the first patch oligonucleotide under stringent conditions while the first patch oligonucleotide is hybridized to the DNA fragments, said first adapter comprising a first primer binding site.
  • FIG. 1 An exemplary schematic of steps to produce cDNA specifically from the RNA genome of a pathogen for use in some embodiments of the method.
  • FIG. 2 An exemplary schematic of steps to anneal selected cDNA with patch oligonucleotides for use in some embodiments of the method.
  • FIG. 2 discloses SEQ ID NOS 551-554, respectively, in order of appearance.
  • FIG. 3 An exemplary schematic of steps to ligate adapter oligonucleotides to concatenate universal primer binding sites to the cDNA, and amplification of the same with universal primers for use in some embodiments of the method.
  • FIG. 4 An exemplary schematic of a massive parallel sequencing protocol for use in some embodiments of the method.
  • FIG. 5 A restriction map of the genome of SARS-CoV-2.
  • the information at the foregoing URL is incorporated by reference in its entirety.
  • FIG. 6 A restriction map of the genome of SARS-CoV-2 (top) and a map of some potential patch oligonucleotides that have been designed capture restriction fragments thus formed, also found at the URL mentioned above for FIG. 5.
  • FIG. 7 A flowchart of an embodiment of a method that creates a cDNA library from a viral RNA genome.
  • the cDNA molecules each include a sample barcode indicating the patient or sample from which the RNA was reverse transcribed and a primer binding site for subsequent PCR amplification.
  • FIG. 8 A flowchart of an embodiment of a method that amplifies pooled cDNA from many subjects made in the embodiment of the method shown in FIG. 7.
  • cDNA including sample barcodes and a common PCR binding site are ligated to an adapter having another PCR binding site and a barcode indicating the identity of the pool.
  • FIG. 9 An embodiment of a control RNA used to validate the test.
  • FIG. 9 discloses SEQ ID NO: 555.
  • FIG. 10 Example of ACTB sequence alignment spanning exons.
  • BA-K064-7296-7345 represents sequence alignment for the genomic DNA captured in this region.
  • FIG. 11 Example of RPP30 amplicon sequence alignment.
  • (Top) RPP-LP & RPP-RP indicate the regions of Patch alignment to the RPP30 gene with the RPP-K064 representing 50 bases of sequencing from a sample.
  • FIG. 11 discloses SEQ ID NOS 556-557, and 557, respectively, in order of appearance.
  • FIG. 12 Example design of a synthetic RNA control.
  • FIG. 12 discloses SEQ ID NO: 555.
  • first”, “second”, and the like are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be termed a second feature or element, and similarly, a second feature or element discussed below could be termed a first feature or element without departing from the teachings of the present disclosure.
  • administering or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient's body externally or internally (or both). It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self- administer a substance is an act of administration.
  • prevention refers to a course of action (such as administering a compound or pharmaceutical composition of the present disclosure) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition.
  • preventing and suppressing need not be absolute to be useful. It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self- administer a substance is an act of prevention.
  • treatment refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition.
  • Such treating need not be absolute to be useful. It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self- administer a substance is an act of treatment.
  • the term "individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.
  • nucleotides refer to any such known groups, natural or synthetic. It includes conventional DNA or RNA bases (A, G, C, T, U), base analogs, e.g., inosine, 5-nitroindazole and others, imidazole-4-carboxamide, pyrimidine or purine derivatives, e.g., modified pyrimidine base 6H,8H- 3,4-dihydropyrimido[4,5-c][1 ,2]oxazin-7-one (sometimes designated “P” base that binds A or G) and modified purine base N6-methoxy-2,6-diaminopurine (sometimes designated "K” base that binds C or T), hypoxanthine, N-4-methyl deoxyguanosine, 4-ethyl-2'-deoxycytidine, 4,6-difluorobenzimidazole and 2,4- difluorobenzene nucleoside analogues, pyren
  • polynucleotide refers to a multimeric compound comprising nucleotides linked together to form a polymer, including conventional RNA, DNA, LNA, BNA, copolymers of any of the foregoing, and analogs thereof.
  • nucleic acid refers to a single stranded polynucleotide or a duplex of two polynucleotides. Such duplexes need not be annealed at all locations, and may contain gaps or overhangs.
  • nick refers to a discontinuity in a double stranded nucleic acid molecule where there is no phosphodiester bond between adjacent nucleotides of one strand.
  • Nucleic acids are "complementary” to each other, as used herein, when a nucleotide sequence in one strand of a nucleic acid, due to orientation of its nucleotide hydrogen atoms, hydrogen bonds to another sequence on an opposing nucleic acid strand (of course, a strand of a nucleic acid may be self- complementary as well).
  • the complementary bases typically are, in DNA, A with T, and C with G, and, in RNA, C with G, and U with A.
  • Complementarity can be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing.
  • “Substantial” or “sufficient” complementary means that a sequence in one strand is not perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex at a given set of hybridization conditions (e.g., salt concentration and temperature).
  • Such conditions can be predicted by using the sequences and standard models to predict the T m of hybridized strands, or by empirical determination of T m by using established methods.
  • T m refers to the temperature at which a population of hybridization complexes formed between two nucleic acid strands are 50% denatured. At a temperature below the T m , formation of a hybridization complex is favored, whereas at a temperature above the Tm, melting or separation of the strands in the hybridization complex is favored.
  • Complementarity may also be defined by the ability of one nucleotide sequence to hybridize with another nucleotide sequence at a given level of stringency. Such stringency is based on the T m of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, 152, Academic Press, San Diego Calif.).
  • the T m of an annealed duplex depends on the base composition of the duplex, the frequency of base mismatches, and the ionic strength of the reaction medium.
  • the T m of a duplex can be calculated by those of ordinary skill in the art based on these two factors using accepted algorithms.
  • Maximum stringency typically occurs at about 5° C below T m ; high stringency at about 5-10° C below T m ; intermediate stringency at about 10-20° C below T m ; and low stringency at about 20-25° C below T m .
  • a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related sequences.
  • stringent by itself in this context refers to intermediate stringency. Terms such as maximally stringent, highly stringent, and poorly stringent, refer to conditions of maximal stringency, high stringency, and low stringency respectively.
  • any given elements of the disclosed embodiments of the invention may be embodied in a single structure, a single step, a single substance, or the like.
  • a given element of the disclosed embodiment may be embodied in multiple structures, steps, substances, or the like.
  • next-gen sequencing can be used to detect biomarkers (such as pathogen genes) in the sample at extremely low initial concentrations. Because the method generates sequence data for one or more RNA biomarkers, it does not depend on perfect complementarity between the biomarker and an oligonucleotide probe, instead allowing the detection of mutant variations of the target. Furthermore, next-gen sequencing can rapidly sequence dozens or hundreds of targets, enabling the sequencing of large amounts of RNA, such as an entire pathogen genome. This ensures that biomarkers can be detected even if they contain mutations or deletions.
  • the biomarker When the biomarker is a group of pathogen genes, they can still be detected even if some of the pathogen genes contain mutations or are deleted.
  • the method may be used to detect a single target sequence or multiple target sequences . Some embodiments of the method comprise detecting at least 10, 20, 30, 40, 50, 100, or 150 biomarkers, such sequences from the pathogen.
  • Other possible RNA biomarkers include mRNA translated from the subject's genome (such as a marker of genetic disease or other disease state), mRNA translated from a pathogen's genome, RNA that is part or all of the genome of a pathogen, miRNA translated from the subject or a pathogen, and siRNA translated from the subject or a pathogen.
  • Samples of a medical nature may be collected from any fluid, tissue, cells, or other bodily material that is likely to contain RNA or the pathogen in question or its genetic material.
  • fluids that could be sampled include saliva, blood, urine, tears, lymph, mucus, semen, vaginal fluid, bile, cerebrospinal fluid, amniotic fluid, synovial fluid, aqueous humor, breast milk, and any combination of two or more of the foregoing.
  • the sample is saliva.
  • Some samples may be mixed, containing two or more of fluid, tissue, and cells. For example, samples collected by a saline mouth rinse would be expected to contain saliva and endothelial cells.
  • Environmental samples and industrial samples may be collected from soil, water, air, equipment, surfaces, or other materials suspected to harbor organisms or RNA.
  • each sample is partially processed before it is mixed into a pool of other samples.
  • the samples may be pooled prior to processing or with only minimal processing.
  • a portion of the sample collected from each individual is processed and/or added to the pooled sample, while a second portion is retained. The retained portion may be used for later confirmatory testing or other purposes.
  • Samples may be taken from a relatively large number of sources, such as individual subjects.
  • samples from over 32 individuals are collected and pooled (either before or during processing).
  • methods samples from over 64 individuals are pooled.
  • at least 95 individuals are pooled.
  • methods samples are pooled from at least 100, 150, 200, 250, 300, 400, or 500 individuals.
  • DNA may be generated from RNA in the sample if RNA is to be analyzed. If a pathogen is to be detected that uses RNA as its genetic material, the method may comprise creating a cDNA library from an individual sample or of a pooled sample suspected of harboring the pathogen.
  • the cDNA library may be constructed using a reverse transcriptase in combination with one or more pairs of reverse transcription primers ("rt primers”).
  • the rt primers are oligonucleotides that complement a region of the RNA, such as a pathogen's genome.
  • random primer pairs may be used to non- specifically create cDNA from the RNA in the sample.
  • one or more pairs of rt primers are used that specifically complement known regions in the targeted RNA, such as known regions in a pathogen's genome.
  • This approach has the advantage of preferably generating cDNA from targeted RNA, instead of from all of the RNA in the sample.
  • a complementary DNA strand to the newly synthesized DNA can then be generated by a DNA polymerase.
  • the parent RNA strand can be displaced by any suitable means, such a denaturation, the use of RNAse, and/or the use of a DNA polymerase that displaces RNA. This results in double-stranded (DS) cDNA.
  • rt primer will hybridize to a sequence in the RNA under conditions of low stringency, moderate stringency, high stringency, or maximum stringency.
  • Multiple primer pairs may be used that hybridize to multiple loci in the pathogen genome (often in multiple overlapping fragments).
  • primer pairs may be used that effectively flank the entire pathogen genome.
  • the rt primers are specific for loci in the genome of SARS-CoV-2. Such primers specific for SARS-CoV-2 may be selected from the below list in Table 1. Note that like-named primers designated "LEFT” and "RIGHT” may be used together as primer pairs.
  • Some embodiments of the method employ one or more primer pairs that are italicized in Table 1.
  • one or more of the primer pairs from Table 1 may be used.
  • the primer pairs may be selected from one or more of the underlined primers in Table 1.
  • the method comprises hybridizing the RNA in the sample with at least 5, 10, 25, 50, 75, 100, 109 125, or 150 primers; in still further embodiments at least 5, 10, 25, 50, 75, 100, or 109 primers are selected from Table 1. Further embodiments of the primers share less than 100% sequence identity with the primers in Table 1.
  • the primers may have at least 50%, 60%, 70%, 80&, 85%, 90%, 95%, 96%, or 97% sequence identity with one or more of the primers listed in Table 1 .
  • Primer pairs may be selected to prime reverse transcription of a portion of the pathogen's genome or the entire pathogen genome.
  • RT-PCR is performed with at least one left primer selected from SEQ ID NO: 11, 17, 19, 29, 35, 36, 43, 44, 51 , 52, 61, 63, 65, 79, 85, 99, 100, 117, 129, 141, 143, 153, 155, 163, 169, 170, 183, 193, 195, and 201.
  • RT-PCR is performed left primers comprising SEQ ID NO: 11 , 17, 19, 29, 35, 36, 43, 44, 51, 52, 61 , 63, 65, 79, 85, 99, 100, 117, 129, 141, 143, 153, 155, 163, 169, 170, 183, 193, 195, and 201.
  • the paired right primer is also used as shown in Table 1.
  • all the primer pairs listed in Table 1 are used, which should result in reverse transcription of the entire SARS-CoV-2 genome.
  • reverse transcription may be performed with a left primer from Table 1 and a standard right primer, such as a P7 primer (which comprises CAA GCA GAA GAC GGC ATA CGA GAT (SEQ ID NO: 550))
  • a standard right primer such as a P7 primer (which comprises CAA GCA GAA GAC GGC ATA CGA GAT (SEQ ID NO: 550))
  • Some embodiments of the method involve concatenating the complement of the target RNA with one or both of a sample barcode identifying the sample and a universal PCR primer binding site.
  • barcode here refers to a nucleotide sequence that can be used to distinguish RNA from a certain sample from others.
  • the sample barcode indicates an individual subject from whom the sample was collected.
  • the barcode may be about 4, 5, 6, 7, 8, 9, 10, or more bases in length. In a preferred embodiment, the barcode may be about 8 bases.
  • Some embodiments of the barcode are located "internal” to the universal primer binding site, such that the barcode will be maintained in the PCR product.
  • RT primers comprises the barcode or the universal PCR binding site (or both) at the 5' end of the molecule.
  • the RT primer will have a sequence capable of selectively hybridizing with a target sequence present in the RNA at or near the 3' end.
  • This recognition sequence will be designed to selectively hybridize with the target RNA under RT-PCR conditions, such as stringent or highly stringent conditions.
  • the recognition sequence may be, for example, any of the sequences from Tables 1 and 2, particularly if the pathogen of interest is SARS-CoV-2.
  • the recognition sequence may share a level of sequence identity of less than 100% with any of the sequences from Tables 1 and 2; for example, the level of sequence identity may be at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • Embodiments of the method that involve the addition a sample barcode to the cDNA have the advantage of identifying which samples in a group of samples have pathogen biomarkers, even if the cDNA is later pooled for mass sequencing. Time and expense are reduced by allowing the immediate identification of the samples that test positive without further testing.
  • Embodiments of the method thethat involve the addition of the universal PCR binding site to the cDNA simplify processing of the pooled cDNA. Such embodiments find particular utility when a population of individual subjects is being screened for a target RNA (such as from a pathogen).
  • a digest may then be constructed of the pooled cDNA or genomic (or other target) DNA.
  • the cDNA libraries may be pooled prior to digest construction.
  • a digest is then made from the cDNA library (when the pathogen has an RNA genome) or from DNA from the sample.
  • the DNA may be isolated or further purified prior to this step.
  • a "digest” refers to a population of nucleic acid fragments from the genome in question with defined ends.
  • defined ends refers to a nucleic acid sequence where both the 5' and 3' end of the sequence is known. In some embodiments of the method at least three, four, five, six, seven, or more than seven bases of the sequence are known.
  • methods for creating defined ends may include restriction endonuclease digestion, single strand specific exonuclease degradation, CRISPR/CAS9, amplification (such as multiplex amplification), or triplex formation and cleavage.
  • the cDNA strands may be modified to add only one defined end. This is because one end of the cDNA strands will already be defined as the sample barcode, primer binding site, or both. This can be accomplished using various methods, such as the methods for creating defined ends described above. In a specific embodiment of the method a single restriction endonuclease is introduced to make a cut at a target cut site and produce the digest.
  • restriction endonuclease enzymes may be used to create nucleic acid sequences with defined ends.
  • Suitable restriction endonuclease enzymes may include type I, type II, type III or type IV restriction endonuclease enzymes.
  • the restriction enzyme used should have recognition sites that flank, and not bisect, the desired nucleic acid sequence.
  • the restriction endonuclease enzymes may be type I restriction endonuclease enzymes.
  • Type I restriction endonuclease enzymes may include Cfrl, Eco377l, EcoAI, EcoDXXI, EcoKI, Eco124l, KpnAI, Hpy4CHv, Ddel, and StySPI.
  • the restriction endonuclease enzymes may be type II restriction endonuclease enzymes.
  • Type II restriction endonuclease enzymes suitable for the methods may be a restriction endonuclease enzyme of type MB, type HE, type IIF, type IIG, type MM, type IIS, or type IIT.
  • Type III restriction endonuclease enzymes may be suitable for the methods.
  • the restriction endonuclease enzymes may be Type IIS restriction endonuclease enzymes.
  • Type IIS restriction endonuclease enzymes may include Fokl, Hgal, Ecil, BceAI, Bbvl, BtgZI, BsmFI, Bpml, and Bsgl.
  • restriction endonuclease enzyme cut sites may be used to define the ends of nucleic acid templates.
  • Components of the restriction enzyme reaction mixture may include the nucleic acid sequence to be digested (e.g., pathogen genome or cDNA library), one or more restriction endonucleases, and salts and buffers essential for optimal activity of the enzymes in the reaction.
  • oligonucleotides may be used to direct Type Ms restriction enzymes to cut at specific sites in the nucleic acid template. This is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a guide for digestion by the type I Is restriction endonuclease enzyme.
  • components of the restriction enzyme reaction may include the nucleic acid sequence to be digested, one or more restriction endonucleases, the oligonucleotides directing the restriction endonuclease cut sites (described below), and salts and buffers essential for optimal activity of the enzymes in the reaction.
  • the upstream and downstream restriction enzyme-directing oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described above.
  • the 5' ends of the upstream restriction enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequence, and may be concatenated at the 3' end of the oligonucleotides to double-stranded nucleotide sequences containing type I Is restriction recognition sites.
  • the 3' ends of the downstream restriction-enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequences, and may be concatenated at the 5' end of the oligonucleotides to double-stranded nucleotide sequences containing type I Is restriction recognition sites.
  • Annealing of the restriction enzyme-directing oligonucleotides to the nucleic acid templates may generally be performed before addition of the restriction enzyme for digestion.
  • annealing reactions may generally contain about 1 pM to about 500 nM of each restriction enzyme-directing oligonucleotide, and about 0.01 to about 0.9% Tween80.
  • annealing of the restriction enzyme-directing oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the restriction enzyme-directing oligonucleotides to target nucleic acid sequences.
  • the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C.
  • the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C.
  • the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more.
  • the annealing temperatures may be about 25, 26, 27, 28, 29,-30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4950, 51, or 52° C.
  • the type I Is restriction enzyme may be added.
  • Double stranded specific restriction enzyme digestion of nucleic acid templates identified by locus- specific oligonucleotides may be used to define ends of the nucleic acid template. This is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve to direct digestion by the double strand specific restriction enzymes.
  • Components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section l(a) above), one or more double stranded specific exonuclease enzymes (described below), the oligonucleotides identifying the nucleic acid template (described below), and salts and buffers essential for optimal activity of the restriction enzymes in the reaction.
  • Non-limiting examples of double stranded specific restriction enzymes suitable for the methods may be exonuclease VII, exonuclease III, exonuclease I, RecJ exonuclease, or Terminator(TM) 5'- Phosphate-Dependent Exonuclease (Epicentre Biotechnologies).
  • the upstream and downstream oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described above.
  • Annealing of the identifying oligonucleotides to the nucleic acid templates may generally be performed before addition of the restriction enzymes.
  • annealing reactions may generally contain about 1 pM to about 500 nM of each oligonucleotide.
  • annealing of the oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the protecting oligonucleotides to target loci. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C.
  • the melting temperature may be about 90, 91 , 92, 93, 94, 95, 96, 97 or 98° C.
  • the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more.
  • the annealing temperatures may be about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 4950, 51 , or 52° C.
  • the denaturing reactions may be incubated at the melting temperature for about 5 to about 30 minutes. In a preferred embodiment, the denaturing reactions may be incubated at the melting temperature for about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes. In some embodiments, the annealing reactions may be incubated at the annealing temperature for about 1 to about 10 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes. After annealing of the identifying oligonucleotides, the restriction enzymes may be added for digestion.
  • Some embodiments of the method comprise creating defined ends using the clustered regularly interspaced short palindromic repeats (CRISPR) approach.
  • CRISPR utilizes the DNA endonuclease CRISPER associated protein 9 (CAS9) to make double stranded breaks in genomic DNA.
  • CAS9 is conjugated with a strand of guide RNA (gRNA), which targets a specific DNA sequence that then becomes the site of the endonuclease.
  • gRNA will generally be at least 20 base pairs in length. Although it may be longer, longer gRNA does not appear to improve specificity.
  • double- stranded breaks can be made in the DNA or cDNA at specific sequences, which become the defined ends.
  • Some embodiments of the method comprise creating defined ends using a multiplex PCR reaction using primer pairs for desired targets on the nucleic acid template.
  • Components of the multiplex PCR amplification reaction may include the nucleic acid sequence to be amplified (e.g., from the pathogen genome), one or more primer pairs for delineating the target nucleic acid sequence on the template to be amplified (described below), one or more nucleotide polymerases (described below), deoxynucleotides, and salts and buffers essential for optimal activity of the polymerases in the reaction.
  • Oligonucleotide PCR primers may be typically synthesized using the four naturally occurring deoxynucleotides dATP, dTTP, dCTP and dGTP. Oligonucleotide primers may also incorporate natural or synthetic deoxynucleotide analogs not normally present in DNA. Incorporation of nucleotide analogs allows for the oligonucleotide primers to be selectively removed after amplification of the target nucleic acid. In some embodiments of the method, a primer may be used such that, at one or more positions of the primer, one or more of the four deoxyribonucleotides in the primer may be replaced with one or more nucleotide analogs.
  • primers may have one of the deoxynucleotides replaced with a nucleotide analog.
  • 25%, 30%, 35% 40%, 50%, 60%, 70%, 80%, 90% or 100% of either dATP, dTTP, dCTP or dGTP in the primers may be replaced with a nucleotide analog.
  • the nucleotide analog may be at the 3'-terminus of the primer.
  • PCR primers may be designed using standard primer design computer software techniques known to individuals skilled in the art.
  • the variables considered during PCR primer design may include primer length, GC pair content, melting temperature, and size of the target nucleic acid amplified by the primer pair.
  • the primers do not form hairpin structures or self- or hetero-primer pairs.
  • primers may comprise a sequence of 15, 20, 25, 30, 35, 40, 45, 50 or more bases complementary to a portion of a template.
  • the primer melting temperature may be 50, 55, 60, 65, 70 or 75° C.
  • the primer melting temperature may be 61, 62, 63, 64, 65, 66 or 67° C.
  • the melting temperature of each primer of the primer pair may be the same. In another embodiment, the melting temperature of each primer of the primer pair may be different for each primer. In yet another embodiment, the difference in melting temperatures between each primer of the primer pair may be 1, 2, 3, 4, 5, 6, 7, 8, 9° C, or more. In another preferred embodiment, the maximum difference in melting temperature between primer pairs may be 5° C. In a preferred embodiment, the GC content of primer may be 10, 20, 30, 40, 50, 60, 70 or 80%. In yet another preferred embodiment the primer pair may be designed to amplify nucleic acid target products that may be 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or more base pairs in length.
  • the nucleotide polymerase may be a DNA polymerase.
  • the nucleotide polymerase may be a thermostable polymerase.
  • the nucleotide polymerase may be a thermostable DNA polymerase.
  • a thermostable polymerase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each PCR cycle.
  • thermostable polymerases may include polymerases isolated from the thermophilic bacteria Thermus aquaticus (Taq polymerase), Thermus thermophilus (Tth polymerase), Thermococcus litoralis (Tli or VENTTM polymerase), Pyrococcus furiosus (Pfu or DEEPVENTTM polymerase), Pyrococcus woosii (Pwo polymerase), other Pyrococcus spp., Bacillus stearothermophilus (Bst polymerase), Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum (Tac polymerase), Thermus rubber (Tru polymerase), Thermus brockianus (DYNAZYMETM polymerase), Thermotoga neapolitana (Tne polymerase), Thermotoga maritime (Tma).
  • the PCR reaction may contain more than one thermostable polymerase enzyme with complementary properties leading to more efficient amplification of target sequences.
  • a nucleotide polymerase with high processivity the ability to copy large nucleotide segments
  • another nucleotide polymerase with proofreading capabilities the ability to correct mistakes during elongation of target nucleic acid sequence
  • the thermostable polymerase may be used in its wild type form.
  • the polymerase may be modified to contain a fragment of the enzyme or to contain a mutation that provides beneficial properties to facilitate the PCR reaction.
  • the thermostable polymerase may be Taq polymerase.
  • Many variants of Taq polymerase with enhanced properties are known and include AmpliTaq(TM), AmpliTaq(TM) Stoffel fragment, SuperTaq(TM), SuperTaq(TM) plus, LA Taq(TM) LApro Taq(TM), and EX Taq(TM).
  • the thermostable polymerase used in the multiplex amplification reaction is the AmpliTaq Stoffel fragment.
  • PCR buffers may generally contain about 10-50 mM Tris-HCI pH 8.3, up to about 70 mM KCI, about 1.5 mM or higher MgCl.sub.2, to about 50-200 mM each of dATP, dCTP, dGTP and dTTP, gelatin or BSA to about 100 pg/ml, and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 at about 0.05- 0.10% v/v.
  • betaine may be added to the PCR reactions at about 0.25 to about 1 M.
  • the multiplex PCR reaction may contain 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more primer pairs. Not all primer pairs will amplify targets with the same efficiency. In some embodiments, PCR primer pairs with similar amplification efficiency may be pooled in separate multiplex PCR reactions to have better representation of all targets. These PCR reactions may be combined after amplification.
  • PCR amplification may be performed at a uniform temperature (isothermal PCR).
  • isothermal PCR methods may include the ramification amplifying method and the helicase-dependent amplification method.
  • PCR amplification may be by thermal cycling between a high temperature to melt the nucleic acid strands, a lower temperature to anneal the primers to the target nucleic acid, and an intermediate temperature compatible with the nucleic acid polymerase to elongate the nucleic acid sequence.
  • the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C.
  • the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C.
  • the annealing temperatures may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C. or more.
  • the annealing temperature may be 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 6970, 71, or 72° C
  • the elongation temperature may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C or more.
  • the elongation temperature may be 70, 71, 72, 73, 74, 75, 80° C or more.
  • the PCR reaction may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the melting temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the annealing temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the annealing temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the elongation temperature for about 1 to about 10 minutes.
  • the PCR reaction may be incubated at the elongation temperature for about 6 minutes. In some embodiments, the PCR reaction is pre-incubated at the melting temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes before cycling between the melting, annealing and elongation temperatures. In a preferred embodiment, the PCR reaction may be pre-incubated at the melting temperature for about 2 minutes.
  • the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more times. In a preferred embodiment, the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.
  • the amplified targets from the PCR reaction described above may be trimmed so the ends of the target regions become internal to the PCR primer sequences.
  • the extent of the trimming may generally be defined by synthetic nucleotide analogs incorporated into the primer pairs described above.
  • oligonucleotides containing 5-bromodeoxyuridine (BdUR) or 5- bromodeoxycytidine (BrdC) may be used as the primers.
  • Primers containing BdUR may be degraded upon exposure to light.
  • the deoxyinosine may be incorporated into primers.
  • Primers containing deoxyinosine may be degraded using Endonuclease V, an enzyme that recognizes and cleaves the sugar phosphate backbone at the deoxyinosine residue.
  • the base of the synthetic nucleotide is first specifically removed, leaving an apurinic or apyrimidinic site (AP site) and an intact sugar-phosphate backbone.
  • the sugar-phosphate backbone is then cleaved at the AP site, generating a nick in the target, which dictates the nucleic acid sequence to be removed by exonuclease enzymes.
  • the base of the synthetic nucleotide analog is removed with a DNA glycosylase enzyme.
  • DNA glycosylases are a family of enzymes that can remove the base of some nucleotide analogs.
  • nucleotide analogs that may be incorporated into primers and that are substrates for glycosylase enzymes may include deoxyuridine, deoxy-7-methylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3-methyladenosine, deoxyinosine, 5-methyl-deoxycytidine, 0-6-methyl-deoxyguanosine, 5-iodo-deoxyuridine, 8-oxy- deoxyguanine, and 1,N 6 -ethenoadenine.
  • Glycosylase enzymes that remove bases from nucleotide analogs incorporated into target nucleic acid sequences may include uracyl DNA glycosylase, 7-methylguanine- DNA glycosylase, 5,6-dihydroxythymidine glycosylase, 3-methyladenine glycosylase, hypoxanthine DNA N- glycosylases, 8-oxoguanine-DNA glycosylase, and alkylpurine-DNA-N-glycosylase.
  • the nucleotide analog may be deoxyuridine.
  • the DNA glycosylase enzyme may be uracil DNA glycosylase.
  • treatments that cleave AP sites may include, but are not limited to, heat, alkaline hydrolysis, tripeptides such as Lys-Trp-Lys and Lys-Tyr-Lys, AP endonucleases such as endonuclease III, endonuclease IV, endonuclease VI, endonuclease VIII, phage T4 UV endonuclease, and the like.
  • the treatment is endonuclease VIII.
  • the resulting single strand overhanging nucleic acid sequence at the 3' termini may be removed using an enzyme with a 3' to 5' single stranded exonuclease activity as depicted in the diagram above.
  • Commonly used 3' to 5' exonucleases that remove single stranded nucleic acids may include exonuclease I and exonuclease VII.
  • the exonuclease is exonuclease I.
  • the unincorporated nucleotides may be removed using enzymes such as apyrase, an ATP diphosphohydrolase that catalyzes the removal of the gamma phosphate from ATP and the beta phosphate from ADP.
  • enzymes such as apyrase, an ATP diphosphohydrolase that catalyzes the removal of the gamma phosphate from ATP and the beta phosphate from ADP.
  • triplex nucleic acid structures may be used to create defined ends of a nucleic acid sequence.
  • Triplex DNA structures may be induced at specific loci by incubating nucleic acid templates with locus-specific oligonucleotides that have been coated with the recombination protein. The triplex structure then produces a single stranded region of nucleic acid available for cleavage by single strand specific endonucleases.
  • components of the restriction enzyme reaction may include the nucleic acid sequence to be digested, one or more recombination proteins, the recombination protein-coated locus-specific oligonucleotides, the endonuclease proteins, and salts and buffers for optimal activity of the enzymes.
  • recombination proteins may include RecA of Escherichia coli, or any homologous recombination protein capable of inducing formation of triplex DNA structure.
  • Non-limiting examples of single strand specific endonucleases may include S1 and BAL1 endonucleases.
  • the resulting digest will contain DNA fragments from the pathogen genome and in many cases DNA fragments from other sources, such as the host or non-target microorganisms and viruses. These DNA fragments will be prepared for multiplex PCR using a "patch” method. This is facilitated using one or more patch oligonucleotides, which serve as a patch between the target sequence and a universal primer binding site to be ligated.
  • a single patch oligonucleotide may be used; in such cases the patch oligonucleotide will bind at the end of the fragment opposite to the first universal PCR primer binding site.
  • the DNA fragments do not initially contain a universal PCR primer binding site, one or more pairs of upstream and downstream patch oligonucleotides may be used that anneal upstream and downstream of one or more target nucleic acid sequences (such as a marker of the pathogen).
  • the universal primer binding sites can then be used during primer-initiated polymerization reactions, such as PCR, to amplify only the target sequences using a small number of universal primers.
  • each universal primer binding site is located on an "adapter” oligonucleotide, which comprises a patch binding site that hybridizes to the patch oligonucleotide under suitable conditions.
  • the patch binding site and the universal primer binding site may overlap or be the same site.
  • the adapter consists of the universal primer binding site.
  • patch ligation reaction mixtures contain the target sequences (e.g., in a pathogen genome digest), the upstream and downstream adapters to be ligated, the upstream and downstream patch oligonucleotides to guide the specific ligation of the adapters, and the enzymes and other components needed for the ligation reaction.
  • target sequences may be nucleic acid sequences with defined ends as described above.
  • the upstream and downstream universal primer binding sites on the adapters may be designed using primer length, GC pair content, and melting temperature criteria.
  • the downstream universal primer binding site may be modified to facilitate further steps.
  • the downstream adapter in an adapter pair may be modified with a 5' phosphate group to enable ligation of the adapter to the amplicon.
  • At least one end of one or more of the adapters may be modified for protection against exonuclease digestion with an exonuclease resistant group.
  • the 3' end of the downstream or upstream adapter may be modified for protection against exonuclease digestion with an exonuclease resistant group.
  • Exonuclease resistant groups may be introduced at the time of synthesis or after synthesis through various chemical means. Modifications may be 3' terminal or slightly internal to the 3' end.
  • nucleic acid sequences exonuclease resistant include, but are not limited to, locked nucleic acids (LNA's), 3'-linked amino groups, 3' phosphorylation, the use of a 3'-terminal cap (e.g., 3'-aminopropyl modification or by using a 3'-3' terminal linkage), phosphorothioate modifications, the use of attachment chemistry or linker modification such as Digoxigenin NHS Ester, Cholesteryl-TEG, biotinylation, thiol modifications, or addition of various fluorescent dyes and spacers such as C3 spacer.
  • the adapter is protected from exonuclease digestion by a C3 spacer.
  • the adapter is protected from exonuclease digestion by a thiol group.
  • no exonuclease resistant group is introduced on the adapter.
  • the reverse transcription primer is joined to one or both of a barcode and a universal PCR binding site as described above, and the exonuclease resistant group is omitted from the adapter.
  • the adapter may be coupled to nucleic acid sequence identification code (barcodes).
  • the nucleic acid barcode may be about 4, 5, 6, 7, 8, 9, 10, or more bases in length. In a preferred embodiment, the nucleic acid barcode may be about 8 bases.
  • the barcode may serve as an indicator of the source of the sample, or other pertinent information about the assay.
  • Some embodiments of the barcode are located "internal” to the universal primer binding site, such that the barcode will be maintained in the PCR product. In some embodiments of the method the barcode will identify a population from which the initial sample was pooled (a "pool barcode”).
  • one or more patch oligonucleotides may be specifically designed for the RNA to be detected, such as RNA from the pathogen. In further embodiments these may take the form of one or more pairs of upstream and downstream patch oligonucleotides.
  • the 5' ends of the upstream patch oligonucleotides may be complementary to one or more target sequences, and may be concatenated to upstream nucleotide sequences complementary to the upstream universal primer sequence on the 3' end.
  • the 3' ends of the downstream patch oligonucleotides may be complementary to downstream sequences, and may be concatenated to nucleotide sequences complementary to the downstream universal primer sequence on the 5' end.
  • patch oligonucleotides are used, but not both.
  • a universal primer binding site is concatenated to the pathogen DNA during the reverse transcription step
  • Further embodiments of the patch may lack 100% complementarity to a sequence in the target RNA, but will specifically hybridize to a sequence in the target RNA. Such specific hybridization may occur under suitable conditions for PCR, or as described in a PCR protocol.
  • Some embodiments of the rt primer will hybridize to a sequence in the target RNA under conditions of low stringency, moderate stringency, high stringency, or maximum stringency.
  • the pathogen is SARS-CoV-2
  • the method uses one or more patch oligonucleotides complementary to portions of the SARS-CoV-2 genome.
  • pairs of left and right patch oligonucleotides are used that are selected from Tables 2 and 3.
  • Right and left patch oligonucleotides with the same name may be preferably used together as a pair.
  • at least 10 pairs of left and right patch oligonucleotides are used that are selected from Tables 2 and 3.
  • at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 162 pairs of left and right patch oligonucleotides are used that are selected from Tables 2 and 3.
  • Some embodiments of the patch oligonucleotides have less than 100% sequence identity with the patch oligonucleotides listed in Tables 2 and 3. Some such embodiments of the patch comprise a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with any of those listed in Tables 2 and 3.
  • only left or right patches are used, selected from Tables 2 and 3 respectively.
  • at least 10 left or right patch oligonucleotides are used selected from Tables 2 and 3.
  • at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 162 left or right patch oligonucleotides are used that are selected from Tables 2 and 3.
  • Some embodiments of the patch have less than 100% sequence identity with the patch oligonucleotide listed in Tables 2 and 3.
  • Some such embodiments of the patch comprise a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with any of those listed in Tables 2 and 3.
  • only left patch oligonucleotides are used.
  • left patch oligonucleotides are used containing sequences at least 75% identical to one or more of SEQ ID NOS: 224, 226, 227, 231, 233, 236, 239, 243, 244, 245, 252, 255, 262, 268, 274, 279, 280, 286, 287, 291, 294, 300, 305, 306, and 308.
  • left patch oligonucleotides are used containing sequences at least 75% identical to five or more of the foregoing. In still more specific embodiments of the method, left patch oligonucleotides are used containing sequences at least 75% identical to ten or more of the foregoing. In still more specific embodiments of the method, left patch oligonucleotides are used containing sequences at least 75% identical to fifteen or more of the foregoing. In still more specific embodiments of the method, left patch oligonucleotides are used containing sequences at least 75% identical to twenty or more of the foregoing.
  • left patch oligonucleotides are used containing sequences at least 75% identical to all of the foregoing. Higher levels of sequence identify may be present, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity.
  • the universal primers may be ligated to nucleic acid sequences.
  • multiple cycles of heating and cooling may be used to melt the target nucleic acid sequence, anneal the patch and adapters, and ligate the adapters to target nucleic acid sequences.
  • the adapter may be ligated to the target nucleic acid using a DNA ligase.
  • the ligase may be theromostable.
  • the ligase is a thermostable DNA ligase.
  • a thermostable DNA ligase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each ligation cycle.
  • Non-limiting examples of theromostable DNA ligases may include HiFi Ligase®, Ampligase® Thermostable DNA Ligase, Taq DNA Ligase from Thermus aquaticus, Tfi DNA ligase from Thermus filiformis, Tth DNA ligase from Thermus thermophilus, Thermo DNA ligase, Pfu DNA ligase from Pyrococcus furiosus, and thermostable DNA ligase from Aquifex pyrophilus.
  • the thermostable polymerase may be used in its wild type form, modified to contain a fragment of the enzyme, or to contain a mutation that provides beneficial properties to facilitate the ligation reaction.
  • the thermostable ligase is HiFi Ligase®
  • Ligation reactions may generally contain about 1 pM to about 500 nM of each patch oligo, about 1 pM to about 500 nM of each adapter, about 3, 4, 5, 6, 7, or 8 units of HiFi Ligase®, and 1x HiFi Ligase Reaction Buffer.
  • ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a sequence of 1, 2, 3, 4 or 5 lower temperatures to anneal the patch oligonucleotides to the target nucleic acid, and a temperature compatible with the ligase to ligate the nucleic acid sequence.
  • ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a first lower temperature to anneal the patch oligonucleotides to the target nucleic acid, a second lower temperature to anneal the adapters to the patch oligonucleotides, and a temperature compatible with the ligase to ligate the nucleic acid sequence.
  • the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C.
  • the melting temperature may be about 90, 91 , 92, 93, 94, 95, 96, 97 or 98° C.
  • the patch oligonucleotide annealing temperatures may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C or more. In a preferred embodiment, the patch oligonucleotide annealing temperatures may be about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 , or 72° C. In another embodiment, the ligation temperature may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C. or more. In a preferred embodiment, the ligation temperature may be about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70° C or more.
  • the ligation reactions may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the ligation reactions may be incubated at the melting temperature for about 30 seconds. In some embodiments, the ligation reactions may be incubated at the patch oligonucleotide annealing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes. In a preferred embodiment, the reactions may be incubated at the patch oligonucleotide annealing temperature for about 2 minutes. In some embodiments, the ligation reactions may be incubated at the universal primer annealing temperature for about 30 seconds to about 5 minutes.
  • the ligation reactions may be incubated at the adapter annealing temperature for about 1 minute. In some embodiments, the ligation reactions may be incubated at the ligation temperature for about 30 seconds to about 5 minutes. In a preferred embodiment, the ligation reactions may be incubated at the ligation temperature for about 1 minute. In some embodiments, the reactions may be pre-incubated at the melting temperature for about 5, 6, 7, 8, 9, 10, 15, 20 or 25 minutes before cycling between the melting, annealing and ligation temperatures. In a preferred embodiment, the ligation reactions may be pre- incubated at the melting temperature for about 15 minutes.
  • the ligation reactions may be cycled between the melting, annealing and ligation temperatures about 10, 50, 100, 150, 200 or more times. In a preferred embodiment, the ligation reactions may be cycled between the melting, ligation, and annealing temperatures about 100 times.
  • exonucleases may be added to the ligation reaction at the completion of the reaction to degrade mis-priming products of the multiplex PCR reaction or non-target DNA (such as residual DNA from the host genome or the genomes of non-target microorganisms present in the sample).
  • the exonucleases may be 3' to 5' exonucleases.
  • Exonucleases may be single stranded or double stranded exonucleases.
  • Non-limiting examples of exonucleases suitable for this step of the reaction may include exonuclease I, exonuclease III and mung bean nuclease. One or more exonucleases may be added.
  • the exonucleases may be exonuclease I and III.
  • This step is particularly preferred in embodiments of the method in which one or more adapters comprise a nuclease-resistant group, as it results in the selective degradation of polynucleotides that are not ligated to the adapter. Additional purification can be performed to separate the adapter-ligated target polynucleotides from excess nucleotides, excess patch oligonucleotides, excess adapter, exonuclease, and other reagents prior to universal amplification. Any suitable separation method can be used.
  • the exonuclease is not added to the ligation reaction.
  • the reverse transcription primer is joined to one or both of a barcode and a universal PCR binding site as described above, and the exonuclease resistant group is omitted from the adapter, exonuclease is not added to the ligation reaction.
  • the products of ligation can be subsequently amplified using PCR with one or more pairs of universal primers, which are complementary to the universal primer sequences.
  • the PCR primers may be coupled to nucleic acid sequences for sequencing.
  • the primers for the final universal PCR may be tailed to lllumina sequencing primers P5 and P7 (lllumina, Inc., San Diego, CA).
  • the primers for the PCR amplification may be complementary to the upstream and downstream universal primer nucleotide sequences ligated.
  • the universal primers hybridize with the primer binding site(s) on the adapters.
  • PCR conditions can be optimized based on the melting temperature of the universal primer and the melting temperature of the template sequences, as described above.
  • the universal PCR reaction mixture may comprise a target sequence ligated to an adapter, a DNA polymerase, a universal primer, and nucleotides (dNTPs).
  • the reaction mixture may further comprise buffers and salts as necessary to modulate desired melting and hybridization temperatures. Variations of the universal PCR reactions may be as described in Section 3 above. Because the universal PCR reaction may be performed with as few as one universal primer, highly efficient amplification can be achieved even if a wide diversity of target sequences are being amplified.
  • the resultant PCR product may be purified prior to sequencing by various suitable methods. In a preferred embodiment the PCR product is purified by magnetic bead technology, such as the AMPURE system (Beckman Coulter, Indianapolis, IN).
  • nucleic acid samples may be sequenced.
  • the nucleic acids sequenced may be the amplicons prepared as described above.
  • Sequencing techniques suitable for the method may be high throughput.
  • High throughput sequencing techniques may include techniques based on chain termination, pyrosequencing (sequence by synthesis), or sequencing by ligation.
  • the lllumina technique is used.
  • high throughput sequencing techniques like true single molecule sequencing (tSMS) may not require amplification of target nucleotide sequences.
  • sequencing may be performed using high throughput sequencing techniques that involve in vitro clonal amplification of the target nucleotide sequence.
  • Non-limiting examples of high throughput sequencing techniques that involve amplification may include solid-phase PCR in polyacrylamide gels, emulsion PCR, rolling-circle amplification, bridge PCR, BEAMing (beads, emulsions, amplification and magnetics)-based cloning on beads, massively parallel signature sequencing (MPSS) to generate clonal bead arrays.
  • the amplicons may be sequenced using PCR techniques as exemplified by llluminaTM sequencing.
  • nucleic acid sequences amplified in the PCR reactions of more than one sample may be pooled for parallel sequencing of nucleic acids prepared in multiple samples. In some embodiments, about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more samples may be pooled for sequencing.
  • sequence data can then be analyzed for the presence of known target sequences, such as genomic sequences from the pathogen.
  • known target sequences such as genomic sequences from the pathogen.
  • genomic sequences such as genomic sequences from the pathogen.
  • Such mutations will not result in false negative results, because the sequence data will contain at least some known canonical genome sequences of the pathogen if it is present, and because most of the mutated sequences would still be expected to be identical to a canonical sequence or known variant thereof.
  • the method is simultaneously highly specific and highly tolerant of deviations from previously known genomic data.
  • sequencing can be performed on multiple pools of DNA fragments simultaneously. For example, when pool barcodes have been used to identify DNA fragments derived from corresponding populations, the DNA from multiple pools or populations may be sequenced together. A positive result can be attributed to the given pool or population based on the barcode. The same is true of the use of sample barcodes. Sequencing may be performed on DNA from multiple individuals (optionally part of multiple pools) simultaneously, and a positive result can be attributed to a single sample or individual based on the sample barcode. C. METHOD OF CONTAGION CONTROL IN A POPULATION
  • a method of controlling contagion of a pathogen in a population comprises performing any of the screening assays described above on a pooled sample from multiple individuals in the population, detecting the pathogen in the pooled sample, and implementing a contagion control measure on one or more individuals in the population.
  • implementing a contagion control measure include acts such as prescribing, instructing, coordinating, or undertaking a measure intended to affect the rate of spread of the pathogen. It is specifically contemplated that instructions or a prescription by a medical professional or public health professional to another person to coordinate, order, or otherwise undertake such a measure is an act of implementation.
  • the presence of the pool barcode sequence can be used to indicate that one or more individuals in the population associated with the pool barcode are positive for the pathogen biomarker. This can be particularly useful when sequencing is performed on multiple pools simultaneously. If a positive result is found based on sequence data, the pool can be identified using the pool barcode.
  • the contagion control measure may involve follow-up testing of one or more individuals who contributed to the pooled sample. For example, a positive result for the pooled sample could lead to individual testing of some or all of the individuals in the pool for the pathogen. Alternatively, the individuals in the pool could be divided into smaller pools and pooled testing could be repeated on the smaller pools.
  • Any individuals confirmed as positive for the pathogen could be isolated from the rest of the population or treated. Such isolation could take the form of quarantine, social distancing requirements, the wearing of protective clothing that prevents spread of the pathogen from the infected individual, the modification of the infected person's working area or living area to prevent contagion, etc.
  • Some embodiments of the method may comprise isolating all persons in the pool, which has the advantage of not requiring follow-up testing. Further embodiments of the method may involve follow-up testing on sub-pools, followed by isolation measures for persons in any sub-pools that test positive.
  • the response to the positive test on the pooled sample is enacting isolation measures for the whole population.
  • the whole population may be subject to a treatment or preventative measure, such as but not limited to: administration of a therapeutic agent, administration of a preventative agent (such as a vaccine), and administration of supportive care.
  • the response to the positive test on the pooled sample is contact tracing. Contact tracing may be performed for all individuals in the pool, all individuals in a sub-pool that tests positive, or all individuals who test positive in individual testing.
  • the response to the positive test is the administration of a treatment for the pathogen to one or more subjects. If the positive test results from testing a pooled population without the use of a sample barcode, the treatment may be administered to the whole population. Alternatively, the treatment may be administered after follow-up testing to a confirmed individual or subgroup of individuals testing positive. If a sample barcode is used in screening the treatment may be administered to the individual associated with the sample barcode.
  • the pathogen is SARS-CoV-2
  • the treatment is a treatment of SARS-CoV-2 disease (i.e., COVID-19) such as one or more of convalescent plasma, an antibody that recognizes SARS-CoV-2, dexamethasone, and remdesivir.
  • Kits are provided for performing the methods described above.
  • the kit comprises reagents for hybridizing a patch oligonucleotide to a target sequence, one or more patch oligonucleotides, reagents for hybridizing an adapter to the patch oligonucleotide, one or more adapters, reagents for ligating the adapter to the target sequence, reagents for performing PCR on the target sequences ligated to the adapter, and a universal primer.
  • Such reagents, patches, adapters, and universal primers may be any that would be suitable for use with the method.
  • a specific embodiment of the kit for detecting SARS-COV-2 comprises one or more patches from Tables 2 and 3.
  • kits comprise one or more pairs of patch oligonucleotides in which one of the pair is selected from Table 2 and the corresponding patch is selected from Table 3.
  • Some embodiments of the kit contains at least 10 pairs of left and right patch oligonucleotide pairs that are selected from Tables 2 and 3. Further embodiments of the kit contain at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 162 pairs of left and right patch oligonucleotide pairs that are selected from Tables 2 and 3.
  • Some embodiments of the patch have less than 100% sequence identity with the patch oligonucleotide listed in Tables 2 and 3.
  • Some such embodiments of the patch comprise a sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with any of those listed in Tables 2 and 3.
  • kit may contain unpaired patch oligonucleotides, for example either left patch oligonucleotides or right patch oligonucleotides.
  • kits only left patch oligonucleotides are used. Some embodiments of the kit contain at least 10 left patch oligonucleotides that are selected from Tables 2. Further embodiments of the kit contain at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 162 left patch oligonucleotides selected from Table 2.
  • left patch oligonucleotides are used containing sequences at least 75% identical to one or more of SEQ ID NOS: 224, 226, 227, 231, 233, 236, 239, 243, 244, 245, 252, 255, 262, 268, 274, 279, 280, 286, 287, 291, 294, 300, 305, 306, and 308.
  • left patch oligonucleotides are used containing sequences at least 75% identical to five or more of the foregoing.
  • left patch oligonucleotides are used containing sequences at least 75% identical to ten or more of the foregoing.
  • left patch oligonucleotides are used containing sequences at least 75% identical to fifteen or more of the foregoing. In still more specific embodiments of the kit, left patch oligonucleotides are used containing sequences at least 75% identical to twenty or more of the foregoing. In still more specific embodiments of the kit, left patch oligonucleotides are used containing sequences at least 75% identical to all of the foregoing. Higher levels of sequence identify may be present, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity. Further embodiments of the kit may contain unpaired patch oligonucleotides, for example either left patch oligonucleotides or right patch oligonucleotides.
  • Kits for use with the method when the pathogen is an RNA virus, viroid, or otherwise has RNA as its genetic material may further comprise reagents for RT-PCR and one or more rt primers that are specific to the pathogen.
  • the RT-PCR reagents may be any that would be suitable for performing the method above.
  • the rt primers in the kit may be selected to recognize sequences from the pathogen's genome.
  • the rt primers are specific to the pathogen's genome.
  • one or more of the rt primers are selected from Table 1.
  • the kit comprises 5, 10, 25, 50, 75, 100, 109 125, or 150 primers.
  • kits comprise at least 5, 10, 25, 50, 75, 100, or 109 primers selected from Table 1. Still further embodiments of the kit comprise one or more rt primers with less than 100% sequence identity to one or more primers in Table 1; in further such embodiments any of the rt primers may have a level of sequence identity with a primer from Table 1 that is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 99.9%. Primer pairs may be selected to prime reverse transcription of a portion of the pathogen's genome or the entire pathogen genome. In a specific embodiment of the kit all the primers listed in Table 1 are used, which should result in reverse transcription of the entire SARS-CoV-2 genome.
  • PROPHETIC EXAMPLE SARS-COV-2 SCREENING USING POOL BARCODES
  • saline salt + water
  • each batch receives a batch identifier and is scanned into a tracking system. 4.
  • Each batch is then processed for enrichment of the viral genome: a. a portion of each batch sample is combined into a combined batch (CB-95).
  • the sample is additionally comprised of human cells and microbes; b.
  • CB-95 undergoes RT-PCR for the conversion of the viral RNA genome to DNA using the rt primers in Table 1
  • region specific primers convert portions of the viral RNA genome to single stranded DNA (cDNA) then on to multiple copies of double stranded DNA (CB-95-cDNA); c.
  • CB-95-cDNA is then subjected to genetic enrichment that results in (i) increases in representation of viral DNA that is present, (ii) addition of adapters used later in the process and (iii) removes non- enriched background DNA (i.e. human, microbial, other virus); i. CB-95-cDNA is digested by two restriction enzymes (Ddel & HpyCH4V); ii. the double digested CB-95-cDNA then enters the patch ligation reaction using the patches in Table 2 (the patches enable the specific addition of adapters possessing universal primer sequences to both ends of each specified portion of the viral genome.
  • one adapter possess chemical modifications that result in the specified viral sequences being resistant to exonuclease digestion); iii. the CB-95-cDNA is subjected to digestion with two exonucleases, serving to degrade contamination by the human and microbial DNA in the sample collection; iv. magnetic beads are then utilized to remove the enzymes and degraded product from the CB-95- cDNA; v. viral specific CB-95-cDNA now undergoes PCR amplification utilizing the universal primer sequences in the adapters with the addition of batch specific DNA barcodes (this process results in the increased abundance of viral specific DNA (CB-95-cDNA-Enriched)); vi. magnetic beads are then utilized to remove enzymes, nucleotides and any other material.
  • the resulting sequences undergo demultiplexing, an analytical process to separate each combined batch's reads, followed by assessment of quality, alignment of reads to the SARS-COV-2 genome and produce result data.
  • Each combined batch is reported to the supplying organization as a listing of batches containing individuals: a. a batch reported as ‘clear’ indicates that all individuals in the batch are virus-free; b. a batch reported as ‘virus detected' indicates that one or more of the individuals in the batch is likely positive for the presence of SARS-COV-2 - individuals that are presumptively positive are identifiable at this point; c. a batch reported as ‘inconclusive’ indicates that the samples did not produce data that is reportable due to quantity, quality or other issue.
  • Batches that contain a ‘virus detected' indication are then processed for individual identification in the following process: a. individual saline samples from the presumptive positive batch (p-CB-95) are transferred into a 96- well microtiter plate; b. the arrayed p-CB-95 samples undergo RT-PCR for the conversion of the viral RNA genome to cDNA; c. p-CB-95-DNA is then subjected to genetic enrichment that results in (i) increases in representation of viral DNA that is present, (ii) addition of adapters used later in the process, (iii) addition of a unique sample identifier and (iv) removes non-enriched background DNA (i.e. human, microbial, other virus).
  • the enriched CB-95 is then prepared for sequencing: a. 95 p-CB-95-DNA-Enriched products are combined together and loaded into a next-generation sequencing flowcell; b. the sequencing cartridge and flowcell are loaded into a sequencer with a runtime of x hours [determined by cycle number & index reads],
  • the resulting sequences undergo an analytical process to separate each combined batch's reads, assess quality, align reads to the SARS-COV-2 genome and produce result data.
  • a report is issued to the supplying organization as a listing of individuals that have been identified as: a. ‘clear’ indicates that this individual is virus-free; b. ‘virus detected' indicates that this individual is likely positive for the presence of SARS-COV-2; c. ‘inconclusive’ indicates that the sample did not produce data that is reportable due to quantity, quality or other issue.
  • PROPHETIC EXAMPLE SARS-COV-2 SCREENING USING SAMPLE BARCODES
  • a protocol is planned (“DirectID”) that does not require an RNA extraction, instead it utilizes a direct RT-PCR protocol that includes the addition of sample specific molecular barcodes on the cDNA. These barcoded samples are then combined to form a batch that proceeds through "ViralPatch” enrichment and sequencing. Individuals that are positive for SARS-CoV-2 are identified following sequence analysis. This method also reduces the use of limited reagents and has the additional advantage of also reducing the turnaround time for individual results.
  • Saline samples are received into the clinical laboratory, accessioned and heat inactivated. The appropriate number of samples for a batch are then scanned and loaded onto a Hamilton Starlet liquid handling system.
  • the Starlet transfers a portion of each saline sample into a different microtiter plate location and combined with RT-PCR master mix and RT-PCR primers.
  • the right primers are a combination of RT primer, sample specific barcode and P7.
  • the left contains only the RT primer sequence. In both primers, the RT sequences are a subset of the Artie primers utilized in BatchID.
  • the regions converted into cDNA will possess a barcode and P7 sequence, allowing multiple samples to be combined for processing together.
  • the Segments are then added to a universal PCR master mix containing a combination of P5 and P7 primers. Post-cycling, the universal PCR product is bead cleaned. The batch is then loaded onto an lllumina MiSeqTM with the following run parameters: read 1: 50 cycles, index 1: 8 cycles, index 2: 8 cycles (dual indexing) or 12 cycles (UMI). To date, all designs have been completed and reagents produced for initial testing.
  • sRNA synthetic RNAs
  • Three synthetic RNAs are designed for addition to each batch. Three regions of the SARS-CoV-2 genome were condensed to the elements needed for RT-PCR, digestion by the restriction enzymes and patching like the companion regions of the SARS-CoV-2 genome.
  • the sRNA carry an unique eight (8) base sequence not found in the SARS-CoV-2 genome, and are seen within the 50 cycles of read 1. Alignment and identification of multiple sRNA barcodes at specified read depths are required for a batch to be considered for reporting. This approach to process controls is shown in FIG. 9.
  • sRNA controls were incorporated into a batch of 100 samples and did not impact the assay's performance in capturing SARS-CoV-2 genomic locations.
  • SARS-CoV-2 was successfully identified while hundreds of copies of one or more of the sRNA controls were identified. Work has been performed to identify the optimal amount of sRNA to add to batch and individual samples.
  • beta-actin ACTB
  • RPP30 ribonuclease P protein subunit p30
  • NTC non-template control
  • the yields of the first preparation were low (reviewed on a gel).
  • an additional human library previously sequenced in the laboratory was added. This added DNA provided the majority of sequencing data resulting in minimal SARS-CoV-2 representation.
  • RNA samples 62 CoV-2 sequences were produced covering 15 regions of the genome that were sequenced 2 or more times.
  • Inactivated virus produced 219 SARS-CoV-2 sequences covering 6 regions of the genome that were sequenced 2 or more times. Individual samples were identified by the unique sample barcode incorporated during the individual RT-PCR step.
  • SARS-CoV-2 was re-sequenced without the addition of the human library.
  • RNA samples 178 CoV-2 sequences were produced covering 38 regions of the genome that were sequenced 2 or more times.
  • Inactivated virus produced 838 SARS-CoV-2 sequences covering 171 regions of the genome that were sequenced 2 or more times.
  • Individual samples were identified by the unique sample barcode incorporated during the individual RT-PCR step.
  • RT-PCR reverse transcription polymerase chain reaction
  • the library yields were higher (as determined by gel analysis) for the samples containing virus.
  • 1,122,767 CoV-2 sequences were produced covering 131 regions of the genome that were sequenced 2 or more times.
  • Inactivated virus produced 3,324 SARS-CoV-2 sequences covering 79 regions of the genome that were sequenced 2 or more times.
  • the clinical sample (SR-38) produced 837 SARS-CoV-2 sequences covering 40 regions of the genome that were sequenced 2 or more times. Sequencing results are shown in Table 5.
  • the same sample set was prepared again with the addition of RNA controls and Proteinase K treatment to remove proteins contained in the samples.
  • the library yields were substantial (as determined by gel analysis) for the samples containing virus.
  • RNA samples 417,392 reads went to RNA controls, 157,736 CoV-2 sequences were produced covering 70 regions of the genome that were sequenced 2 or more times. Inactivated virus produced 1,137,000 reads for the RNA controls, 6,585 SARS-CoV-2 sequences covering 59 regions of the genome that were sequenced 2 or more times. And, the clinical sample (SR-38) produced 266,106 RNA control reads and 157 SARS-CoV-2 sequences covering 10 regions of the genome that were sequenced 2 or more times. The sequencing results are shown in Table 6.
  • the assay is a targeted capture and next generation sequencing test for SARS-CoV-2.
  • the SARS-CoV-2 primer and capture protocol is designed to detect RNA from the SARS-CoV-2 in respiratory specimens from patients as recommended for testing by public health authority guidelines. Samples are collected utilizing the Kailos Genetics ViraWashTM Collection Kit [5mL of saline, instructions for gargling and rinsing of the oral cavity] and pooled into groups of 25 for sequencing (Assure Sentinel). Utilizing the same process, individual tests are performed on samples comprising positive pools.
  • RNA extraction The RNA is then extracted from the c-batch utilizing the KingFisher Flex and MagMAX Viral/Pathogen RNA isolation reagents. Subsequently, three (3) synthetic RNAs for Process Controls are added;
  • RT-PCR - To the c-batch, a combination of 109 CoV-2 RT-PCR primers pairs are added. These create 98 overlapping regions for reverse transcription and second strand cDNA synthesis. Additional RT- PCR primers are added for Human Beta-actin (ACTB) and Ribonuclease P protein subunit p30 (RPP30).
  • ACTB Human Beta-actin
  • RPP30 Ribonuclease P protein subunit p30
  • the NEB OneTaq One-Step RT-PCR kit and BioRad C1000 Touch Thermocyclers are utilized for the RT reaction. Beckman Coulter AMPureXP magnetic bead based SPRI chemistry or Fisher Scientific Sera-Mag Select are utilized to purify the resulting double stranded cDNA;
  • CoV-Patch Ligation A combination of 162 pairs of CoV-Patch oligonucleotides (see T ables 2 and 3 above), adapters containing PCR primer sites and endonuclease resistance modifications (see the list of viral patch adapters below) are added to the reaction. Additional Patch oligos are added for Human Beta- actin (ACTB) and Ribonuclease P protein subunit p30 (RPP30). Following the addition of DNA Taq ligase, the c-batch undergoes thermocycling in a BioRad C1000 Touch Thermocycler;
  • Exonuclease degradation - Exonucleases I & III are added to the CoV-Patch ligated product and incubated in BioRad C1000 Touch Thermocycler;
  • Bead clean-up - AMPure or Sera-Mag Select magnetic beads are utilized to remove digested products and exchange the buffers;
  • PCR Amplification The purified CoV-Patch ligation product is combined with a DNA Polymerase and universal PCR primers (see the list of viral patch adapters below).
  • the primers are designated Left iIPCR & Right HAPCR i7BC and consist of universal primer sequences and a eight base batch barcode. Components are amplified in a BioRad C1000 Touch Thermocycler;
  • Bead clean-up - AMPure or Sera-Mag Select magnetic beads are utilized to remove digested products and exchange the buffers.
  • Sequencing - Multiple c-batch products are combined into a sequencing pool.
  • the sequencing pool is loaded onto an lllumina flowcell: NextSeq 500/550 v2.5 kits Mid (130M clusters) or High (400M clusters) output kits or MiSeq v2 Reagent Kits (up to 15M reads) followed by sequencing on either a lllumina MiSeq or NextSeq instrument.
  • Sequencing reads are 50 cycles single end followed by two index reads of 8 cycles each.
  • a sequencing run analysis produces BCL files; b. Demultiplexing of BCL files using lllumina bcl2fasq resulting in FastQ files for each sample; c. Alignment of FastQ files to the reference genomes using BWA ALN to create BAM files for each sample.
  • a sample is identified as ‘Negative’ when less than 6 regions of the SARS-CoV-2 genome are sequenced to a depth of 5 or more quality reads (>47 bases).
  • a sample is identified as ‘Positive’ when 6 or more regions of the SARS-CoV-2 genome are sequenced to a depth of 5 or more quality reads (>47 bases).
  • the NEB OneTaq One- Step RT-PCR kit and BioRad C1000 Touch Thermocyclers are utilized for the RT reaction. Beckman Coulter AMPureXP magnetic bead based SPRI chemistry or Fisher Scientific Sera-Mag Select are utilized to purify the resulting double stranded cDNA;
  • CoV-Patch Ligation A combination of 162 pairs of CoV-Patch oligonucleotides (See Tables 2 and 3), adapters containing PCR primer sites and endonuclease resistance modifications (see the list of viral patch adapters below) are added to the reaction. Additional Patch oligos are added for Human Beta-actin (ACTB) and Ribonuclease P protein subunit p30 (RPP30). Following the addition of DNA Taq ligase, the Array undergoes thermocycling in a BioRad C1000 Touch Thermocycler;
  • Exonuclease degradation - Exonucleases I & III are added to the CoV-Patch ligated product and incubated in BioRad C1000 Touch Thermocycler;
  • Bead clean-up - AMPure or Sera-Mag Select magnetic beads are utilized to remove digested products and exchange the buffers;
  • PCR Amplification The purified CoV-Patch ligation product is combined with a DNA Polymerase and universal PCR primers (see the list of viral patch adapters below).
  • the primers are designated Left iIPCR & Right HAPCR i7BC and consist of universal primer sequences and a eight base batch barcode. Components are amplified in a BioRad C1000 Touch Thermocycler;
  • Bead clean-up - AMPure or Sera-Mag Select magnetic beads are utilized to remove digested products and exchange the buffers.
  • Sequencing - Multiple Array products each possessing a differing batch barcode (see the list of viral patch adapters below), are combined into a sequencing pool.
  • the sequencing pool is loaded onto an lllumina flowcell: NextSeq 500/550 v2.5 kits Mid (130M clusters) or High (400M clusters) output kits or MiSeq v2 Reagent Kits (up to 15M reads) followed by sequencing on either a lllumina MiSeq or NextSeq instrument. Sequencing reads are 50 cycles single end followed by two index reads of 8 cycles each.
  • a sequencing run analysis produces BCL files; b. Demultiplexing of BCL files using lllumina bcl2fasq resulting in FastQ files for each sample; c. Alignment of FastQ files to the reference genomes using BWA ALN to create BAM files for each sample. d. Statistics generated utilizing SamTools and BedTools. e. A sample is identified as ‘Negative’ when less than 6 regions of the SARS-CoV-2 genome are sequenced to a depth of 5 or more quality reads (>47 bases). A sample is identified as ‘Positive’ when 6 or more regions of the SARS-CoV-2 genome are sequenced to a depth of 5 or more quality reads (>47 bases).
  • NTC Negative/No Template Control
  • RNA controls are included (Table 14) .
  • the Process controls are designed to regions of the CoV-2 genome that include or are adjacent to regions utilized in the CDC RT-qPCR assays. Regions of the genome bounded by the left and right CoV- Patches have been removed and replaced with a synthetic six base sequence that identifies the resulting sequence of the molecule as a synthetic control. Additional regions between the RT/2nd strand primer and the restriction enzyme sites were removed to enable the length of the molecule to be synthesized.
  • These synthetic RNA molecules undergo the same processing as the viral genome including extraction, reverse transcription, second strand synthesis, restriction digestion, patch ligation, universal PCR, sequencing and analysis.
  • FIG. 12 shows an example design of a synthetic RNA control.
  • This oral wash sample was utilized for serial dilutions that included comparative Ct values of 26, 28, 30, 32, 34, 36, 38 & 40. Each step in the series was prepared in triplicate and each replicate was sequenced independently.
  • RT-PCR Ct value of 40 (upper limit for most RT-PCR assays)
  • a higher threshold was selected to evaluate in the second phase.
  • a single sample with a comparable RT-PCR Ct value of 36 was utilized in the second phase for 20 sequencing replicates, resulting in 100% of samples detecting CoV-2 above quality thresholds (Table 7).
  • the quality threshold for CoV-2 detection is a combination of 6 or more regions of the genome sequenced to a sequencing read depth of 5 or more each.
  • Pathogenic coronavirus genomes were analyzed in silico to determine what portions, if any, of their genomes would be sequenced should their RNA be present in a sample. Utilizing NCBI's BLASTn algorithm to compare the CoV-2 Patch oligonucleotides (see Tables 2 & 3) with the indicated strain identified by a Taxon ID. Details of the alignment are included in Table 8. Alignments indicate that such closely related strains of coronavirus could be captured, sequenced and analyzed when they were present in the processed sample.
  • samples were prepared for SARS-coronaviras HKU1 , NL63 & MERS and sequenced. All controls performed as expected including a positive control for SARS-CoV-2.
  • the SARS-coronavirus HKU1, NL63 & MERS were not detected by the assay, indicating the assay exclusively detects SARS-CoV- 2
  • CoV-2 patches align with portions of some of the genomes, there are additional requirements of (i) restriction sites flanking the patch sights and (ii) two patches, a left and right within 1 kilobase of one another, for the genome region to progress beyond the patch step of the protocol, thus further limiting the potential for these regions to be sequenced.
  • the CoV-2 patch oligos were compared to each genome to show the largest potential set of genomic regions that could contaminate the comparison with the CoV-2 genome.
  • RNA from each sample type was placed in a background matrix of human oral rinse. Each group was assessed in duplicate. All controls performed as expected including a positive control for SARS-CoV-2.
  • Group 5 hMPV, Parainfluenza virus 1, Parainfluenza virus 2
  • Group 6 H1N1, Flu A, Flu B
  • Group 7 RSV, Rhinovirus 16, Rhinovirus 17
  • each patient provided an oral rinse sample for testing with the FRT RT-PCR assay.
  • the FRT testing was completed within 24 hours and the remaining saline sample was stored at -80°C prior to transfer to Kailos Genetics for Assure Sentinel NGS testing.
  • a total of 21 paired NP swab/oral rinse samples were compared, with FRT RT-PCR C t values ranging from 16.8 to 39.8 (Table 10). All three initial methods, FRT RT-PCR and Assure Sentinel NGS detected CoV-2 in patient samples. Detection of CoV-2 in both NP swab and oral rinse samples confirms that the ViraWashTM oral rinse is comparable to NP swab collection and suitable for use in RT-PCR and NGS testing.
  • Table 10 Comparison of samples collected with NP swabs & oral rinse (saline) tests with NGS, FRT RT- PCR and one of three IVDs: Cobas 6800 (C), Gene Xpert (GX), Hologic Panther (P). RT-PCR C t values highlighted in gray are above the level of detection (LOD) of Assure Sentinel reporting.
  • C Cobas 6800
  • GX Gene Xpert
  • P Hologic Panther
  • Oral rinse samples were collected, tested and aliquots of a minimum of 500uL of oral rinse were supplied to for Assure testing.
  • pool #1-3 & #32-38 Three replicates of pool #1-3 & #32-38 were prepared and all remaining pools were prepared as singletons. Following sequencing and quality assessment, each pool was evaluated for the presence or absence of CoV-2. Of the negative pools, thirty-five of thirty-six CoV-2 met the criteria for ‘no detection' (Table 11a). Pool 6 met the criteria for detection of CoV-2 due to the presence of 7 regions of the genome that were sequenced to a depth of 5 or more, triggering the assessment of individual samples for assessment. All 25 samples were tested individually with levels of CoV-2 sequence data that meet the criteria for ‘not detected', therefore all negative samples matched the comparator lab results of ‘not detected'.
  • Table 11a Comparison of single sample RT-PCR and sequencing of negative sample pools
  • Table 6a Comparison of individual SARS-CoV-2-negative sample results using RT-PCR (OraRisk, FRL &
  • Table 12b Comparison of single samples for which SARS-CoV-2 was detected with RT-PCR and Assure Sentinel NGS. Extended times for transport and freeze/thaw cycles may have been factors in detection limit issues in samples with low viral counts (i.e. high Ct values).
  • the patch oligonucleotides from Tables 2 and 3 above may be used.
  • Patch Adapter 1 AAT GAT ACG GCG ACC ACC GAG ATC TAC ACN NNN NNN NNA CAC CGT CTT AGA GAA TGA GGA AGG TGG GGA GT (SEQ ID NO: 546)
  • N describes a unique molecular identifier comprised of twelve (12) random nucleotides Patch Adapter 2: AGT GTG GGA GGG TAG TTG GTG TTT CCA GT * C * A * C (SEQ ID NO: 547)
  • N describes an 8-base batch identifier listed below:
  • Exhibit 1 BLASTn alignments with Coronavirus strains 229E
  • P-CoV2-001-L KF430201 .1 92.857 28 2 0 7784 7811 15998 15971 0.002 42.8 P-CoV2-001-L KF430201 .1 81.579 38 7 0 1682 1719 20751 20714 0.023 40.1 P-CoV2-001-L KF430201 .1 84.848 33 5 0 7902 7934 15246 15214 0.080
  • 38.3 P-CoV2-001-L KF430201 .1 95.652 23 1 0 2720 2742 15271 15249 0.080
  • 38.3 P-CoV2-001-L KF430201 .1 86.207 29 4 0 2533 2561 16480 16452 0.28 35.6 P-CoV2-001-L KF430201.1 88.462 26 3 0 225 250 28734 28709 0.98 34.6 P-CoV2-001-L KF430201 .1 91.304 23 2 0 1584 16
  • P-CoV2-001-L AE015929.1 100.000 20 0 0 3443 3462 400451 400432 7.4 37.4
  • a method of screening for an RNA in a population of samples comprising: (a) creating a cDNA library from a sample containing RNA, using a first 3' adapter oligonucleotide that functions as a reverse transcription primer that will hybridize with a target sequence present in the RNA, wherein the first 3' adapter oligonucleotide comprises: a sample barcode identifying the sample, and a first PCR primer binding site; (b) pooling the cDNA library with a plurality of additional cDNA libraries, to create a pooled cDNA library; (c) creating a cDNA digest of the pooled cDNA library, wherein the cDNA digest comprises cDNA fragments; (d) joining a second adaptor oligonucleotide to the cDNA fragments at the 5' end of said cDNA fragments, said second adapter oligonucleotide comprising: a pool barcode identifying the pool, and a second
  • a method of screening for an RNA in a population of samples comprising: (a) creating a cDNA library by performing RT-PCR on a sample of multiple RNA molecules including a targeted RNA molecule, using a first 3' adapter oligonucleotide that functions as a reverse transcription primer, wherein the first 3' adapter oligonucleotide comprises: (i) a sequence capable of selectively hybridizing with a target sequence present in the targeted RNA molecule, (ii) a sample barcode identifying the sample, and (iii) a first PCR primer binding site; wherein the cDNA library comprises a plurality of double stranded DNA molecules, each comprising the sample barcode and the first PCR primer binding site; (b) pooling the cDNA library with a plurality of additional cDNA libraries, to create a pooled cDNA library; (c) creating a cDNA digest of the pooled cDNA library, wherein the cDNA digest comprises
  • Emb. 3 The method of any one of embodiments 1 and 2, wherein the target sequence originates from a pathogen.
  • Emb. 4 The method of any one of embodiments 1-3, wherein the target sequence originates from a pathogen, and wherein the method is a method of detecting the pathogen.
  • Emb. 5. The method of any one of embodiments 1-4, wherein the sample is from an individual subject.
  • Emb. 6. The method of any one of embodiments 1-5, wherein the sample is from an individual subject and wherein the target RNA originated from a pathogen in the subject.
  • Emb. 7 The method of any one of embodiments 1-6, wherein the sample is from an individual subject and wherein the barcode is unique to the individual subject in the pool.
  • Emb. 8 The method of any one of embodiments 1-7, comprising specifically hybridizing a patch oligonucleotide to the cDNA fragments originating from the RNA at the 5' end of said cDNA fragments prior to joining the second adapter oligonucleotide to the cDNA fragments, and hybridizing the second adaptor oligonucleotide to the patch oligonucleotide while the patch oligonucleotide is hybridized to the cDNA fragments.
  • Emb. 9 The method of any one of embodiments 1-8, wherein multiple 3' adapter oligonucleotides are used in creating the cDNA library, wherein the multiple 3' adapter oligonucleotides each comprise the same sample barcode and the same first PCR primer binding site, wherein the target sequence is present in a targeted RNA molecule from a pathogen, and wherein each of the multiple 3' adapter oligonucleotides comprises a different sequence capable of selectively hybridizing with a different target sequence present in the pathogen.
  • Emb. 10 The method of any one of embodiments 1-9, wherein the target RNA originated from an RNA virus.
  • Emb. 11 The method of any one of embodiments 1-10, wherein the target RNA sequence originated from a pathogen, and wherein the cDNA digest comprises cDNA fragments not originating from the pathogen.
  • Emb. 12 The method of any one of embodiments 1-11, wherein the target sequence is present in a targeted RNA molecule; wherein the cDNA digest comprises cDNA fragments not originating from the targeted RNA molecule; wherein the second adapter oligonucleotide comprises a sequence capable of selectively hybridizing to a patch oligonucleotide; and the patch oligonucleotide does not hybridize with the cDNA fragments not originating from the targeted RNA molecule.
  • the second adapter oligonucleotide comprises a sequence capable of selectively hybridizing to a patch oligonucleotide; wherein the target sequence is present in a targeted RNA molecule; wherein the targeted RNA molecule originates from a pathogen; wherein the cDNA digest comprises cDNA fragments not originating from the pathogen; and wherein the patch oligonucleotide does not hybridize with the cDNA fragments not originating from the pathogen.
  • Emb. 14 The method of any one of embodiments 1-13, wherein the target sequence is present in a targeted RNA molecule; wherein the cDNA library is created using multiple primer pairs each of which is specific to the targeted RNA molecule; and one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 15 The method of any one of embodiments 1-14, wherein the target sequence is present in a targeted RNA molecule; wherein the targeted RNA molecule originates from a pathogen, the cDNA library is created using multiple primer pairs each of which is specific to the pathogen, and one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 16 The method of any one of embodiments 1-15, wherein the target sequence is present in a targeted RNA molecule; wherein the cDNA library is created using at least 50 primer pairs each of which is specific to the targeted RNA molecule; one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 17 The method of any one of embodiments 1-16, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA originates from a pathogen; the cDNA library is created using at least 50 primer pairs each of which is specific to the pathogen; and one of the primers in the primer pair is a portion of the 3' adapter oligonucleotide.
  • Emb. 18 The method of any one of embodiments 1-17, wherein: the target sequence is present in a targeted RNA molecule; and the cDNA library is created using at least 100 primer pairs each of which is specific to the targeted RNA molecule, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 19 The method of any one of embodiments 1-18, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule originates from a pathogen; and the cDNA library is created using at least 100 primer pairs each of which is specific to the pathogen, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 20 The method of any one of embodiments 1-19, wherein: the target sequence is present in a targeted RNA molecule; and the cDNA library is created using at least 109 primer pairs each of which is specific to the targeted RNA molecule, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 21 The method of any one of embodiments 1-20, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule originates from a pathogen; the cDNA library is created using at least 109 primer pairs each of which is specific to the pathogen; and one of the primers in the primer pair is a portion of the 3' adapter oligonucleotide.
  • Emb. 22 The method of any one of embodiments 1-21, wherein the cDNA library is created using one or more primer pairs listed in Table 1, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 23 The method of any one of embodiments 1-22, wherein the cDNA library is created using one or more primer pairs each having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
  • Emb. 24 The method of any one of embodiments 1-23, wherein the cDNA library is created using at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 primer pairs listed in Table 1, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 25 The method of any one of embodiments 1-24, wherein the cDNA library is created using all primer pairs listed in Table 1, one of the primers in the primer pair being a portion of the 3' adapter oligonucleotide.
  • Emb. 26 The method of any one of embodiments 1-25, wherein the second adapter comprises sequence capable of selectively hybridizing to a patch oligonucleotide, and comprising: specifically hybridizing the patch oligonucleotide to the cDNA fragments; and joining the second adapter to the cDNA fragments by hybridizing the second adapter to the patch oligonucleotide.
  • Emb. 27 The method of any one of embodiments 1-26, wherein the target sequence is present in a targeted RNA molecule; comprising: sequencing the target amplicon by massive parallel sequencing; and detecting a sequence corresponding to the targeted RNA molecule.
  • Emb. 28 The method of any one of embodiments 1-27, wherein the target sequence is present in a targeted RNA molecule; comprising: sequencing the target amplicon by massive parallel sequencing; and detecting a sequence corresponding to the targeted RNA molecule on the same strand as the sample barcode.
  • Emb. 29 The method of any one of embodiments 1-28, wherein the target sequence is present in a targeted RNA molecule; comprising digesting the cDNA fragments not originating from the targeted RNA molecule with an exonuclease prior to amplifying the cDNA fragments.
  • Emb. 30 The method of any one of embodiments 1-29, wherein the target sequence is present in a targeted pathogen; comprising digesting the cDNA fragments not originating from the pathogen with an exonuclease prior to amplifying the cDNA fragments.
  • Emb. 31 The method of any one of embodiments 1-30, wherein the target sequence is present in a targeted RNA molecule; the targeted RNA molecule is from a pathogen; and at least 10 pathogen sequences are detected.
  • Emb. 32 The method of any one of embodiments 1-31 , wherein the target sequence is present in a targeted RNA molecule; and wherein at least 10 sequences form the targeted RNA molecule are detected.
  • Emb. 33 The method of any one of embodiments 1-32, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule is from a pathogen; and at least 10 pathogen sequences are detected.
  • Emb. 34 The method of any one of embodiments 1-33, wherein: the target sequence is present in a targeted RNA molecule; and at least 20 sequences form the targeted RNA molecule are detected.
  • Emb. 35 The method of any one of embodiments 1-34, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule is from a pathogen; and at least 20 pathogen sequences are detected.
  • Emb. 36 The method of any one of embodiments 1-35, wherein: the target sequence is present in a targeted RNA molecule; and at least 120 sequences form the targeted RNA molecule are detected.
  • Emb. 37 The method of any one of embodiments 1-36, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule is from a pathogen; and at least 120 pathogen sequences are detected.
  • Emb. 38 The method of any one of embodiments 1-37, wherein: the target sequence is present in a targeted RNA molecule; and patch oligonucleotide hybridizes under stringent conditions to the DNA fragments originating from the targeted RNA molecule.
  • Emb. 39 The method of any one of embodiments 1-38, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule originates from a pathogen; and the patch oligonucleotide hybridizes under stringent conditions to the DNA fragments originating from the pathogen.
  • the target sequence is present in a targeted RNA molecule; the targeted RNA molecule originates from a pathogen; and the patch oligonucleotide hybridizes under stringent conditions to the DNA fragments originating from the pathogen.
  • a method of generating a pathogen-specific amplicon from a population of subjects comprising: (a) creating a DNA digest from a pooled sample of multiple DNA amplification products created from multiple individuals in the population, wherein each of the multiple DNA amplification products comprises a sample barcode identifying the individual and a first PCR primer binding site at a terminal end, and wherein the DNA digest comprises multiple DNA fragments originating from the pathogen; (b) specifically hybridizing a patch oligonucleotide to the DNA fragments originating from the pathogen; (c) hybridizing an adaptor to the patch oligonucleotide while the patch oligonucleotide is hybridized to the DNA fragments, said adapter comprising a (d) second PCR primer binding site; and (e) amplifying the DNA fragments originating from the pathogen using a first primer that hybridizes with the first primer binding site and a second primer that hybridizes with the second primer binding site to create a target amplicon.
  • Emb. 41 The method of embodiment 40, wherein the DNA digest is a cDNA digest.
  • Emb. 42 The method of any one of embodiments 40-41 , comprising generating the DNA digest from a pooled cDNA library; the pooled cDNA library comprising a cDNA library generated from a sample containing RNA and a plurality of additional cDNA libraries.
  • Emb. 43 The method of any one of embodiments 1-42, comprising sequencing the target amplicon by massive parallel sequencing.
  • Emb. 44 The method of any one of embodiments 1-43, comprising sequencing the target amplicon by massive parallel sequencing, and detecting a sequence associated with a pathogen.
  • Emb. 45 The method of any one of embodiments 1-44, comprising sequencing the target amplicon by massive parallel sequencing, and detecting a sequence associated with a pathogen on the same strand as the sample barcode.
  • Emb. 46 The method of any one of embodiments 1-45, wherein the cDNA digest is created using a restriction enzyme.
  • Emb. 47 The method of any one of embodiments 1-46, wherein the cDNA digest is created using at least two restriction enzymes.
  • Emb. 48 The method of any one of embodiments 1-47, wherein the cDNA digest is created using CRISPR.
  • Emb. 49 The method of any one of embodiments 1-48, wherein the cDNA digest is created using PCR to generate a population of amplification products that are the cDNA fragments.
  • Emb. 50 The method of any one of embodiments 1-49, wherein the plurality of additional cDNA libraries are each generated from an individual sample from one of a plurality of subjects in a population. Emb. 51. The method of any one of embodiments 1-50, wherein the plurality of additional cDNA libraries are each generated from an individual sample from one of over 32 subjects in a population.
  • Emb. 52 The method of any one of embodiments 1-51 , wherein the plurality of additional cDNA libraries are each generated from an individual sample from one of over 64 subjects in a population.
  • Emb. 53 The method of any one of embodiments 1-52 , wherein the plurality of additional cDNA libraries are each generated from an individual sample from at least 95 subjects in a population.
  • Emb. 54 The method of any one of embodiments 1-53, wherein the patch oligonucleotide has at least 50% sequence identity with a patch oligonucleotide from Tables 2 and 3.
  • Emb. 55 The method of any one of embodiments 1-54, wherein the patch oligonucleotide has at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with a patch oligonucleotide from Tables 2 and 3.
  • Emb. 56 The method of any one of embodiments 1-55, wherein at the adapter comprises an identification code.
  • Emb. 57 The method of any one of embodiments 1-56, wherein at least one of the adapter comprises an identification code identifying the pooled sample.
  • Emb. 58 The method of any one of embodiments 1-57, comprising implementing a contagion control measure based on the characteristics of the target amplicon.
  • Emb. 59 The method of any one of embodiments 1-58, comprising: sequencing the target amplicon by massive parallel sequencing; detecting a biomarker of a pathogen in the target amplicon; and implementing a contagion control measure based on the presence of the biomarker.
  • Emb. 60 The method of any one of embodiments 1-59, comprising: sequencing the target amplicon by massive parallel sequencing; detecting a biomarker of a pathogen in the target amplicon; detecting the sample barcode on the same strand as the biomarker; and implementing a contagion control measure for an individual associated with the sample barcode based on the presence of the biomarker.
  • Emb. 61 The method of any one of embodiments 1-60, further comprising purifying the target amplicon prior to sequencing the target amplicon.
  • Emb. 62 The method of any one of embodiments 1-61 , comprising separating restriction fragments originating from the targeted sequence from digested DNA.
  • Emb. 63 The method of any one of embodiments 1-62, wherein: the target sequence is present in a targeted RNA molecule; the targeted RNA molecule originates from a pathogen; and the method comprises separating restriction fragments originating from the pathogen from digested non-pathogen DNA.
  • Emb. 64 The method of any one of embodiments 1-63, comprising sequencing multiple target amplicons from multiple pooled samples by massive parallel sequencing.
  • Emb. 65 A method of controlling the contagion of a disease caused by a pathogen, the method comprising: screening a population for the pathogen according to the method of any one of embodiments 40-64, whereby a pathogen sequence is detected by sequencing the amplicon; and wherein the method includes implementing an anti-contagion measure for the population.
  • Emb. 66 The method of controlling the contagion of disease of embodiment 65, wherein the pathogen sequence that is detected is linked to the sample barcode; and comprising implementing the anticontagion measure of an individual associated with the sample barcode.
  • Emb. 67 The method of controlling the contagion of disease of any one of embodiments 65-66, wherein the pathogen sequence that is detected is linked to the sample barcode, and comprising administering a treatment for the pathogen to an individual associated with the sample barcode.
  • Emb. 68 The method of controlling the contagion of disease of any one of embodiments 65-67, wherein the pathogen sequence that is detected is linked to the sample barcode, wherein the pathogen is SARS-CoV-2; and comprising administering a treatment for SARS-CoV-2 to an individual associated with the sample barcode.
  • Emb. 69 The method of controlling the contagion of disease of any one of embodiments 65-68, wherein the pathogen sequence that is detected is linked to the sample barcode, wherein the pathogen is SARS-CoV-2, and comprising administering a treatment for SARS-CoV-2 to an individual associated with the sample barcode, the treatment comprising at least one of: convalescent plasma, an antibody that recognizes SARS-CoV-2, dexamethasone, and remdesivir.
  • Emb. 70 The method of controlling the contagion of disease of any one of embodiments 65-69, further comprising testing each subject for the presence of a biomarker of the pathogen; and detecting the presence of the biomarker in an individual subject; wherein the anti-contagion measure comprises a measure to prevent spread of the pathogen from the individual subject to others in the population.
  • Emb. 71 The method of controlling the contagion of disease of any one of embodiments 65-70, comprising: retaining a portion of the sample for confirmatory testing; detecting a sample barcode on the same strand as the pathogen sequence, wherein the sample barcode is associated with a putatively infected individual who gave the sample; testing the retained portion for the presence of a biomarker of the pathogen; and detecting the presence of the biomarker in the putatively infected individual who gave the sample; wherein the anti-contagion measure comprises a measure to prevent spread of the pathogen from the putatively infected individual to others in the population.
  • Emb. 72 A target amplicon that is a product of the method according to any one of embodiments 1- 64.
  • kits for detection of a pathogen in a sample comprising: (a) a reverse transcription primer comprising a sequence capable of selectively hybridizing with a target sequence present in a pathogen RNA, a sample barcode sequence for identifying an individual test subject, and a first PCR primer binding site; (b) a patch oligonucleotide that hybridizes under stringent conditions to a first genomic sequence from the pathogen; and (c) a first adaptor oligonucleotide configured to hybridize with a first patch oligonucleotide under stringent conditions while the patch oligonucleotide is hybridized to the first genomic sequence from the pathogen, the first adapter oligonucleotide comprising a first primer binding site.
  • Emb. 74 The kit of embodiment 73, comprising one or more of: a reverse transcriptase, a second reverse transcription primer, and a dNTP mixture.
  • Emb. 75 The kit of any one of embodiments 73-74, comprising all of: a reverse transcriptase, a second reverse transcription primer, and a dNTP mixture.
  • Emb. 76 The kit of any one of embodiments 73-75, wherein the reverse transcription primer has at least 70% sequence identity with a primer in Table 1.
  • Emb. 77 The kit of any one of embodiments 73-76, comprising a second reverse transcription primer having at least 70% sequence identity with a primer in T able 1.
  • Emb. 78 The kit of any one of embodiments 73-77, comprising: two rt primers each selected from Table 1.
  • Emb. 79. The kit of any one of embodiments 73-78, wherein the patch oligonucleotide has at least 70% sequence identity with a patch oligonucleotide selected from Table 2 or Table 3.
  • Emb. 80 The kit of any one of embodiments 73-79, wherein the patch oligonucleotide is selected from Table 2 or Table 3.
  • a method of screening for a pathogen in a population of subjects comprising: (a) creating a cDNA library by performing RT-PCR on a pooled sample of multiple individual samples taken from multiple individuals in the population; (b) creating a DNA digest comprising DNA fragments originating from the pathogen; (c) specifically hybridizing a first patch oligonucleotide to the DNA fragments originating from the pathogen; (d) specifically hybridizing a second patch oligonucleotide to the DNA fragments originating from the pathogen; (e) hybridizing a first adaptor to the first patch oligonucleotide while the first patch oligonucleotide is hybridized to the DNA fragments, said first adapter comprising a first primer binding site; (f) hybridizing a second adaptor to the second patch oligonucleotide while the second patch oligonucleotide is hybridized to the DNA fragments, said second adapter comprising a second primer binding site and an
  • Emb. 82 The method of embodiment 81 , wherein the pathogen is an RNA virus.
  • Emb. 83 The method of any one of embodiments 81-82, wherein the DNA digest comprises DNA fragments not originating from the pathogen.
  • Emb. 84 The method of any one of embodiments 81-83, wherein the DNA digest comprises DNA fragments not originating from the pathogen, and wherein neither patch oligonucleotide hybridizes with the DNA fragments not originating from the pathogen.
  • Emb. 85 The method of any one of embodiments 81-84, wherein the cDNA library is created using random primers.
  • Emb. 86 The method of any one of embodiments 81-85, wherein the cDNA library is created using multiple primer pairs each of which is specific to the pathogen.
  • Emb. 87 The method of any one of embodiments 81-86, wherein the cDNA library is created using at least 50 primer pairs each of which is specific to the pathogen.
  • Emb. 88 The method of any one of embodiments 81-87, wherein the cDNA library is created using at least 100 primer pairs each of which is specific to the pathogen.
  • Emb. 89 The method of any one of embodiments 81-88, wherein the cDNA library is created using at least 109 primer pairs each of which is specific to the pathogen.
  • Emb. 90 The method of any one of embodiments 81-89, wherein the cDNA library is created using one or more primer pairs listed in Table 1.
  • Emb. 91 The method of any one of embodiments 81-90, wherein the cDNA library is created using one or more primer pairs each having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a primer listed in Table 1.
  • Emb. 92 The method of any one of embodiments 81-91 , wherein the cDNA library is created using at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 primer pairs listed in Table 1.
  • Emb. 93 The method of any one of embodiments 81-92, wherein the cDNA library is created using all primer pairs listed in T able 1.
  • Emb. 94 The method of any one of embodiments 81-93, the method further comprising: (i) creating a DNA digest from a pooled sample of multiple individual samples taken from multiple individuals in the population, the DNA digest comprising DNA fragments originating from the pathogen and DNA fragments not originating from the pathogen; (j) specifically hybridizing a first patch oligonucleotide to the DNA fragments originating from the pathogen; (k) specifically hybridizing a second patch oligonucleotide to the DNA fragments originating from the pathogen; (I) hybridizing a first adaptor to the first patch oligonucleotide while the first patch oligonucleotide is hybridized to the DNA fragments, said first adapter comprising a first primer binding site; (m) hybridizing a second adaptor to the second patch oligonucleotide while the second patch oligonucleotide is hybridized to the DNA fragments, said second adapter comprising a second primer binding site and an exonucle
  • Emb. 95 The method of any one of embodiments 81-94, comprising creating a first amplicon by amplifying polynucleotides in a pooled sample of multiple individual samples taken multiple individuals in the population, and wherein the DNA digest is a digest of the first amplicon.
  • Emb. 96 The method of any one of embodiments 81-95, comprising separating a nucleic acid fraction from the pooled sample, from which the pathogen amplicon is created.
  • Emb. 97 The method of any one of embodiments 81-96, comprising digesting the DNA fragments not originating from the pathogen with an exonuclease prior to amplifying the DNA fragments, to create digested non-pathogen DNA.
  • Emb. 98 The method of any one of embodiments 81-97, wherein the DNA digest is created using a restriction enzyme.
  • Emb. 99 The method of any one of embodiments 81-98, wherein the DNA digest is created using at least two restriction enzymes.
  • Emb. 100 The method of any one of embodiments 81-99, wherein the DNA digest is created using CRISPR.
  • Emb. 101 The method of any one of embodiments 81-100, wherein the DNA digest is created using PCR to generate a population of amplification products that are the DNA fragments.
  • Emb. 102 The method of any one of embodiments 81-101, comprising collecting the multiple individual samples from the multiple individuals in the population.
  • Emb. 103 The method of any one of embodiments 81-102, comprising creating the pooled sample by combining at least a portion of each of the multiple individual samples.
  • Emb. 104 The method of any one of embodiments 81-103, wherein the pooled sample is from greater than 32 individuals.
  • Emb. 105 The method of any one of embodiments 81-104, wherein the pooled sample is from greater than 64 individuals.
  • Emb. 106 The method of any one of embodiments 81-105, wherein the pooled sample is from at least 95 individuals.
  • Emb. 107 The method of any one of embodiments 81-106, wherein at least 10 pathogen sequences are detected.
  • Emb. 108 The method of any one of embodiments 81-107, wherein at least 20 pathogen sequences are detected.
  • Emb. 109 The method of any one of embodiments 81-108, wherein at least 150 pathogen sequences are detected.
  • Emb. 110 The method of any one of embodiments 81-109, wherein the first and second patch oligonucleotides hybridizes under stringent conditions to the DNA fragments originating from the pathogen.
  • Emb. 111 The method of any one of embodiments 81-110, wherein at least one of the first and second patch oligonucleotides has at least 50% sequence identity with a patch oligonucleotide from Tables 2 and 3.
  • Emb. 112. The method of any one of embodiments 81-111, wherein at least one of the first and second patch oligonucleotides has at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity with a patch oligonucleotide from Tables 2 and 3.
  • Emb. 113 The method of any one of embodiments 81-112, wherein at least one of the first and second adapter comprises an identification code.
  • Emb. 114 The method of any one of embodiments 81-113, wherein the exonuclease resistant group is a locked nucleic acid (LNA), 3'-linked amino, 3' phosphate, a 3'-terminal cap, a 3'-aminopropyl, a 3'-3' terminal linkage, phosphorothioate, attachment chemistry or linker modification, Digoxigenin NHS Ester, Cholesteryl-TEG, biotinylation, thiolation, a fluorescent dye, a spacer, a C3 spacer, or a combination of two or more of the foregoing.
  • LNA locked nucleic acid
  • the exonuclease resistant group is a locked nucleic acid (LNA), 3'-linked amino, 3' phosphate, a 3'-terminal cap, a 3'-aminopropyl, a 3'-3' terminal linkage, phosphorothioate, attachment chemistry or linker modification, Digoxigen
  • Emb. 115 The method of any one of embodiments 81-114, wherein the exonuclease resistant group is a thiol group.
  • Emb. 116 The method of any one of embodiments 81-115, wherein the DNA fragments originating from the pathogen are separated from the digested non-pathogen DNA and the exonuclease using magnetic beads.
  • Emb. 117 The method of any one of embodiments 81-116, wherein at least one of the first and second adapter comprises an identification code identifying the pooled sample.
  • Emb. 118 The method of any one of embodiments 81-117, further comprising purifying the pathogen amplicon prior to sequencing the pathogen amplicon.
  • Emb. 119 The method of any one of embodiments 81-118, comprising separating restriction fragments originating from the pathogen from the digested non-pathogen DNA and from the exonuclease after the digestion step.
  • Emb. 120 The method of any one of embodiments 81-119, comprising sequencing multiple pathogen amplicons from multiple pooled samples by massive parallel sequencing.
  • Emb. 121 The method of any one of embodiments 81-120, whereby a pathogen sequence is detected by sequencing the amplicon; and wherein the method includes implementing an anti-contagion measure for the population.
  • Emb. 122 The method of any one of embodiments 81-121, whereby a pathogen sequence is detected by sequencing the amplicon; and wherein the method includes implementing an anti-contagion measure for the population; the method further comprising testing each subject for the presence of a biomarker of the pathogen; and detecting the presence of the biomarker in an individual subject; wherein the anti-contagion measure comprises a measure to prevent spread of the pathogen from the individual subject to others in the population.
  • Emb. 123 The method of any one of embodiments 81-122, whereby a pathogen sequence is detected by sequencing the amplicon; wherein the method includes implementing an anti-contagion measure for the population; wherein a portion of each of the multiple individual samples was retained for confirmatory testing; wherein each retained portion is tested for the presence of a biomarker of the pathogen; and the method comprising detecting the presence of the biomarker in an individual subject; wherein the anti-contagion measure comprises a measure to prevent spread of the pathogen from the individual subject to others in the population.
  • Emb. 124. A pathogen amplicon that is a product of the method of any one of embodiments 81-123.
  • a kit for detection of a pathogen in a sample comprising: a reverse transcription primer that hybridizes under stringent conditions with a genomic sequence from an RNA pathogen; a first patch oligonucleotide that hybridizes under stringent conditions to a first genomic sequence from the pathogen; a second patch oligonucleotide that hybridizes under stringent conditions to a second genomic sequence from the pathogen; a first adaptor oligonucleotide configured to hybridize with the first patch oligonucleotide under stringent conditions while the first patch oligonucleotide is hybridized to the genomic sequence, said first adapter comprising a first primer binding site; and a second adaptor oligonucleotide configured to hybridize with the second patch oligonucleotide under stringent conditions while the second patch oligonucleotide is hybridized to the genomic sequence, said second adapter comprising a second primer binding site and an exonuclease resistant group
  • the kit of embodiment 125 comprising one or more of: a reverse transcriptase, a reverse transcription primer, and a dNTP mixture.
  • Emb. 127 The kit of any one of embodiments 125-126, comprising all of: a reverse transcriptase, a reverse transcription primer, and a dNTP mixture.
  • Emb. 128 The kit of any one of embodiments 125-127, comprising: two rt primers each having at least 70% sequence identity with a primer in Table 1.
  • kit of any one of embodiments 125-128, comprising: two rt primers each selected from Table 1.
  • Emb. 130 The kit of any one of embodiments 125-129, wherein the first patch oligonucleotide has at least 70% sequence identity with a patch oligonucleotide selected from Table 2 or Table 3; and wherein the second patch oligonucleotide has at least 70% sequence identity with a patch oligonucleotide selected from Table 2 or Table 3.
  • Emb. 131 The kit of any one of embodiments 125-130, wherein the first patch oligonucleotide is selected from Table 2 or Table 3; and wherein the second patch oligonucleotide is selected from Table 2 or Table 3.

Abstract

L'invention concerne un procédé de criblage de grandes populations de sujets en vue de déterminer la présence d'un ARN, tel que l'ARN provenant d'un pathogène. Le procédé combine le regroupement d'échantillons, la RT-PCR ciblée, l'utilisation d'amorces de PCR universelles et le séquençage parallèle massif. Le procédé est extrêmement sensible, et s'il est combiné à un séquençage du génome entier, confère une spécificité extrêmement élevée combinée à une résilience élevée à une variation génétique potentielle chez un pathogène. Le procédé peut être utilisé pour insérer un "code barres" unique qui identifie l'échantillon spécifique (tel qu'un échantillon provenant d'un patient individuel), dans l'ADN produit à partir de l'ARN, ce qui permet à l'ADN provenant de nombreux échantillons d'être regroupé en vue d'un séquençage tout en permettant encore l'identification d'éventuels échantillons individuels dont le test est positif.
PCT/US2021/035240 2020-05-29 2021-06-01 Test d'acide nucléique de masse WO2021243333A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP21813023.5A EP4157300A2 (fr) 2020-05-29 2021-06-01 Test d'acide nucléique de masse

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063032120P 2020-05-29 2020-05-29
US63/032,120 2020-05-29
US202063125501P 2020-12-15 2020-12-15
US63/125,501 2020-12-15

Publications (2)

Publication Number Publication Date
WO2021243333A2 true WO2021243333A2 (fr) 2021-12-02
WO2021243333A3 WO2021243333A3 (fr) 2022-03-03

Family

ID=78722930

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/035240 WO2021243333A2 (fr) 2020-05-29 2021-06-01 Test d'acide nucléique de masse

Country Status (2)

Country Link
EP (1) EP4157300A2 (fr)
WO (1) WO2021243333A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022029157A3 (fr) * 2020-08-06 2022-06-09 F. Hoffmann-La Roche Ag Compositions et procédés pour détecter le coronavirus 2 du syndrome respiratoire aigu sévère (sars-2), de la grippe a et de la grippe b

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10320519A1 (de) * 2003-04-30 2004-11-25 4Base Lab Gmbh Advanced Molecular Analysis Verfahren zum Nachweis infektiöser (+)-Strang-RNA-Viren, insbesondere infektiöser Enteroviren
US11111514B2 (en) * 2008-09-05 2021-09-07 Washington University Method for multiplexed nucleic acid patch polymerase chain reaction
US20120010091A1 (en) * 2009-03-30 2012-01-12 Illumina, Inc. Gene expression analysis in single cells
JP6907202B2 (ja) * 2015-07-31 2021-07-21 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ ウイルス由来の治療剤を解析する方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022029157A3 (fr) * 2020-08-06 2022-06-09 F. Hoffmann-La Roche Ag Compositions et procédés pour détecter le coronavirus 2 du syndrome respiratoire aigu sévère (sars-2), de la grippe a et de la grippe b

Also Published As

Publication number Publication date
WO2021243333A3 (fr) 2022-03-03
EP4157300A2 (fr) 2023-04-05

Similar Documents

Publication Publication Date Title
US10704091B2 (en) Genotyping by next-generation sequencing
US10711269B2 (en) Method for making an asymmetrically-tagged sequencing library
US20180251757A1 (en) Capture Methodologies for Circulating Cell Free DNA
US8741564B2 (en) Quantitative nuclease protection assay (QNPA) and sequencing (QNPS) improvements
US9249456B2 (en) Base specific cleavage of methylation-specific amplification products in combination with mass analysis
CA2892646A1 (fr) Procedes pour analyse genomique ciblee
CN115044645A (zh) Dna文库的高效率构建
CA3149610A1 (fr) Analyse de cellules uniques
JP2011518568A (ja) Ｄｎａに基づくプロファイリングアッセイのための物質及び方法
WO2021243333A2 (fr) Test d'acide nucléique de masse
US20220042120A1 (en) Integrated proviral sequencing assay
US20230129100A1 (en) Detection of low abundance nucleic acids
Stawski Preparing whole genome human mitochondrial DNA libraries for next generation sequencing using Illumina Nextera XT
KR20230124636A (ko) 멀티플렉스 반응에서 표적 서열의 고 감응성 검출을위한 조성물 및 방법
JP2023520590A (ja) 病原体診断検査
CN116348614A (zh) 开关寡核苷酸
US10066262B2 (en) Methods for amplification of nucleic acids utilizing hairpin loop or duplex primers
AU2015203545A1 (en) Quantitative nuclease protection assay (qnpa) and sequencing (qnps) improvements
WO2023034747A1 (fr) Évaluation d'épitranscriptome
CA2904863C (fr) Procede d'amplification d'acides nucleiques cibles fragmentes a l'aide d'une sequence d'assemblement
CA3185142A1 (fr) Methodes d'identification de marqueurs de rejet de greffe
CN111373042A (zh) 用于选择性扩增核酸的寡核苷酸
Campbell Automatable virtual array screening system for rapid analysis of mitochondrial DNA polymorphism

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21813023

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021813023

Country of ref document: EP

Effective date: 20230102

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21813023

Country of ref document: EP

Kind code of ref document: A2