WO2013040060A2 - Acides nucléiques pour détection multiplex du virus de l'hépatite c - Google Patents

Acides nucléiques pour détection multiplex du virus de l'hépatite c Download PDF

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WO2013040060A2
WO2013040060A2 PCT/US2012/054901 US2012054901W WO2013040060A2 WO 2013040060 A2 WO2013040060 A2 WO 2013040060A2 US 2012054901 W US2012054901 W US 2012054901W WO 2013040060 A2 WO2013040060 A2 WO 2013040060A2
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probes
sequence
probe
hcv
collection
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PCT/US2012/054901
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WO2013040060A3 (fr
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Philip Alexander Rolfe
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Pathogenica, Inc.
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    • 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
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/519—Detection characterised by immobilisation to a surface characterised by the capture moiety being a single stranded oligonucleotide
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention is directed to sets of nucleic acid probes for multiplex detection of hepatitis viruses and methods of using the probes.
  • HCV Hepatitis C Virus
  • Embodiments of the present invention include optimized nucleic acid probes, and methods of using them, that enable the skilled artisan to simultaneously detect HCV from a clinical sample, determine the genotype and sub-genotype (or genotypes) present, and detect the presence of a wide variety of mutations known to confer resistance to various direct acting antiviral (DAA) drugs.
  • the invention is based, at least in part, on the discovery of sequences, from sets of large query hepatitis C virus (HCV) sequences such as whole genomes, which can be used in multiplex diagnostic assays that dramatically reduce assay time and cost, compared to conventional diagnostics.
  • HCV hepatitis C virus
  • nucleic acids and methods of the invention enable the skilled artisan to identify hepatitis C virus and differentiate between closely related strains thereof based on the sequence of regions containing, for example, single nucleotide polymorphisms (SNPs), insertions, deletions, or indels (sites where a colocalized insertion and deletion has occurred, resulting in a net gain or loss in nucleotides).
  • SNPs single nucleotide polymorphisms
  • insertions, deletions, or indels sites where a colocalized insertion and deletion has occurred, resulting in a net gain or loss in nucleotides.
  • the nucleic acid probes and methods of the invention may be further multiplexed and used in automated systems, such as microplates, for high throughput processing of large numbers of samples by centralized laboratory, hospital, and/or diagnostic facilities.
  • aspects of the invention provide nucleic acid probes and mixtures comprising a plurality of nucleic acid probes capable of circularizing capture of a region of interest.
  • aspects of the invention include a single-stranded nucleic acid probe comprising a nucleic acid sequence of the formula:
  • a and C are probe-arms designed to hybridize, potentially with some number of mismatches, to a target nucleic acid sequence.
  • a list of probe arms used in this invention is included in tables 1-4.
  • B is a backbone sequence.
  • the backbone sequence B comprises a cleavage site.
  • the cleavage site is a restriction endonuclease recognition site.
  • the backbone sequence B comprises one or more detectable moieties.
  • the one or more detectable moieties are each independently selected from a barcode sequence and a primer-binding sequence.
  • the backbone contains non-Watson-Crick nucleotides, including, for example, abasic furan moieties, and the like.
  • the backbone sequence B includes one or more primer binding sites that can be used to amplify the circularized probe sequence using rolling circle amplification, PCR, or similar techniques.
  • the primer sequences also contain adapters such that the amplification product can be sequenced on a particular next-generation sequencing platform such as the Ion Torrent PGM, Ion Torrent Proton, lllumina MiSeq, lllumina HiSeq, Solid XL, 454 GS, or a nanopore platform.
  • one or more of the primer sequences contains a barcode sequence such that several samples can be amplified each with a unique barcode and the sequencing reads de-multiplexed during analysis.
  • each probe arm sequence in the group consisting of columns 2 and 3 from tables 1 -4 is contained in at least one probe in the plurality of probes.
  • the plurality of probes includes a subset of the probes in tables 1-4, where the probes in the subset have been chosen to detect a specific set of drug resistance mutations or to determine a subset of viral genotypes.
  • the composition comprising a plurality of probes comprises extracted nucleic acids from a test sample.
  • the extracted nucleic acids may be from a biological sample.
  • the biological sample may be from a human patient.
  • the composition comprising a plurality of probes comprises at least one sample internal calibration standard nucleic acid. In some embodiments, the composition comprising a plurality of probes comprises at least one probe that specifically hybridizes with the sample internal calibration standard nucleic acid.
  • the composition comprising a plurality of probes comprises extracted nucleic acids from a test sample.
  • kits comprising the composition comprising a plurality of probes as described herein and instructions for use.
  • the kit may also comprise reagents for obtaining a sample (e.g., swabs), and/or reagents for extracting RNA, and/or enzymes, such as polymerase and/or ligase to capture a region of interest.
  • aspects of the invention include a method of detecting the presence of one or more strains of hepatitis C virus in a test sample, comprising:
  • aspects of the invention also include a method of detecting the genotype of one or more strains of hepatitis C virus (HCV) in a test sample, comprising:
  • a region of interest is captured by polymerase-dependent extension from the 3' terminus of sequence C of a probe in the plurality of probes. In further embodiments, the region of interest is captured by sequence-specific ligation of a linking oligonucleotide.
  • the method of detecting the presence of one or more strains of hepatitis C virus in a test sample or of detecting the genotype of one or more strains of HCV in a test sample includes the step of amplifying the circularized probe to form a plurality of amplicons containing the captured region or regions of interest.
  • the method of detecting the presence of one or more strains of hepatitis virus in a test sample or of detecting the genotype of one or more strains of HCV in a test sample includes the step of treating the mixture with a nuclease to remove linear nucleic acids between the steps of capturing and detecting a region of interest (steps (b) and (c) of each of the methods described above).
  • the method includes the step of linearizing the circularized probe by cleavage with a site-specific endonuclease.
  • the method of detecting the presence of one or more strains of hepatitis C virus in a test sample includes the step of sequencing the region of interest.
  • the method of detecting the presence of one or more strains of hepatitis C virus in a test sample further includes the step of comparing the sequence of the captured region of interest to the sequence of known HCV genomes.
  • the method of detecting the presence of one or more strains of HCV or of determining the genotype of the one or more strains includes the step of comparing the sequence of the captured region to the predicted capture regions of a previously sequenced HCV genomes or partial genomes.
  • the method of detecting the presence of one or more strains of hepatitis C virus in a test sample includes the step of analyzing the sequence of the captured region of interest with respect to the sequence of known hepatitis C virus genomes and a model of sequencing errors to estimate the proportions or abundances of the hepatitis C strains in the test sample.
  • the method of detecting the genotype of one or more strains of HCV in a test sample includes the step of comparing the sequence of the captured region of interest to a database of known HCV mutations.
  • the database of known HCV mutations is a database of known HCV drug resistance mutations.
  • the method of detecting the genotype of one or more strains of HCV in a test sample includes the step of analyzing the sequence of the captured region of interest with respect to the sequence of known HCV genomes and a model of sequencing errors to estimate the proportions or abundances of one or more strains of HCV in a test sample. In some embodiments, the method of detecting the genotype of one or more strains of HCV in a test sample includes the step of analyzing the sequence of the captured region of interest with respect to the sequence of known HCV genomes and mutations and a model of sequencing errors to estimate the proportions or abundances of one or more mutations a test sample.
  • the test sample is obtained from a human subject. In some embodiments, the test sample is blood obtained from a human subject.
  • the method of detecting the presence of one or more strains of hepatitits C virus, of determining the genotype of one or more strains of HCV in a test sample, or detecting mutations in the HCV genome in the test sample includes the step of adding a sample internal calibration standard to the test sample. In some embodiments, the method further comprises the steps of adding a probe that specifically hybridizes with the sample internal calibration standard and detecting the sample internal calibration standard.
  • the method of detecting the presence of one or more strains of hepatitis C virus or of determining the genotype of one or more strains of HCV in a test sample includes the step of formatting the results to inform physician decision making.
  • the formatting includes providing an estimated quantity of one or more HCV genotypes of interest.
  • the formatted results comprise a therapeutic recommendation based on the one or more HCV genotypes detected.
  • the methods of using this invention achieves high specificity in detecting or sequencing only HCV nucleic acid compared to any human nucleic acids that may be present in the sample.
  • the nucleic acids processed by this method are sequenced on a next-generation sequencing machine, less than 50%, 25%, 10%, or 1 % of the resulting sequencing reads or sequencing reads that pass a quality filter represent human nucleic acids. This specificity is advantageous as it reduces the number of sequencing reads required to achieve a desired depth of sequencing for the HCV genome.
  • Figure 1 A schematic diagram of a probe hybridized to the target nucleic acid.
  • the two homologous probe arms are shown, as are the two universal primer binding sites in the backbone that are present in certain embodiments.
  • Figure 2 A schematic of the protocol of an embodiment in which (1 ) probes are hybridized to target nucleic acids in a sample, (2) a polymerase copies the reverse- complement of the target into the probe molecule and a ligase closes the circle, (3) exonuclease enzymes digest away target and unused probe molecules and (4) a pair of adapter-primers amplify circularized probe molecules containing the copied target region, adding a barcode and next-generation sequencing machine adapters in the process.
  • Figure 3 Specific or degenerate primers initiate the reverse-transcription reaction. Each primer contains a homology region to the RNA and a non-binding tail.
  • Panel B shows the content of the tail: a molecule-specific barcode or dogtag followed by a probe binding site. With such a primer and suitably long dogtag, each cDNA molecule will contain a unique dogtag.
  • Panel C shows the capture of the resulting cDNA molecule (or its complement if the first strand cDNA was later amplified in a PCR reaction) by a probe. The probe binds to a target in the reverse-transcribed RNA and to the probe arm binding site such that the molecule-specific barcode will be captured and sequenced.
  • Figure 4 The protocol can be performed in a single tube and the resulting material used with any sequencing platform.
  • Figure 5 The distribution of the 436 probes along the HCV genome.
  • the NS3 gene is between roughly 3kb and 4k, NS5A between roughly 6kb and 7kb, and NS5B from roughly 7kb to 9.5kb.
  • the large number of probes overlapping certain coordinates reflects the enormous genetic diversity between the HCV strains seen in patients around the world. While certain technologies perform poorly on certain strains, the invention described here uses a large set of probes to ensure efficient capture of any sample.
  • Figure 6 Agreement between Sanger Sequencing, another next-generation sequencing approach, and the invention described here (Pathogenica DxSeq) on a subset of clinical samples.
  • Figure 7 Graphical summary of results for the 19 genotype-1a and 11 genotype- l b samples showing the coverage and results of a set of known drug resistance mutations in the NS3, NS5a, and NS5b genes.
  • Figure 8 A list of selected HCV drug resistance mutations and the drugs to which they indicate resistance. Presence of even a small fraction of resistant molecules in a patient sample indicates that the drug should not be prescribed.
  • Figure 9 Agreement in HCV genotype detection between the Versant Genotyping assay and the Pathogenica DxSeq assay disclosed here.
  • Figure 10 The three panes show scatterplots comparing the frequency of alternate alleles detected in an HCV sample between two replicates. Each point represents the frequency of the alternate allele at a single locus in the gene. Because the presence of small numbers of drug resistant viral particles in a patient can predict therapy failure, detection of alternate alleles at low frequency is critical for clinical HCV assays. The invention described here demonstrates a strong correlation between replicates across the entire range of allele frequencies.
  • Figures 11-14 show genotyping results for clinical sample 10 in genes NS3,
  • each column represents a single codon.
  • the vertical coordinate indicates the frequency of the alternate allele at that codon in the clinical sample.
  • the color of the circle indicates whether the alternate allele is a known drug resistance mutation.
  • One aspect of the invention provides mixtures of circularizing "capture” probes suitable for sensitive, rapid, and highly specific detection of one or more hepatitis C viruses in complex samples.
  • Probe refers to a linear, unbranched polynucleic acid comprising two homologous probe sequences separated by a backbone sequence, where the first homologous probe sequence is at a first terminus of the nucleic acid and the second homologous probe sequence is at the second terminus to the nucleic acid, and where the probe is capable of circularizing capture of a region of interest of at least 2 nucleotides.
  • “Circularizing capture” refers to a probe becoming circularized by incorporating the sequence complementary to a region of interest.
  • A is a probe arm sequence listed in column 2 of tables 1 -4;
  • B is a backbone sequence.
  • Figure 1 shows a schematic of a probe hybridized to a target nucleic acid (either RNA or DNA).
  • embodiments encompass a probe, which includes two homologous probe sequences A and C, each of which may specifically hybridize to a different target sequence in a hepatitis viral genome adjacent to a region of interest.
  • a probe may comprise any one of the pairs of homologous probe sequences in columns 2 and 3 of tables 1-4.
  • the probes may further comprise a backbone sequence, which contains a primer binding site between the homologous probe sequences.
  • the homologous probe sequence at the 3' end of the probe is termed the extension arm and the homologous probe sequence at the 5' end of the probe (probe segment A) is termed the ligation or anchor arm.
  • the probe/target duplexes are suitable substrates for polymerase- dependent incorporation of at least two nucleotides on the probe (on the extension arm), and/or ligase-dependent circularization of the probes (either by circularizing a polymerase-extended probe or by sequence-dependent ligation of a linking
  • Capture reaction refers to a process where one or more probes contacted with a test sample has possibly undergone circularizing capture of a region of interest, wherein the first and second homologous probe sequences in the probe have
  • Capture reaction products refers to the mixture of nucleic acids produced by completing a capture reaction with a test sample.
  • Amplification reaction refers to the process of amplifying capture reaction products.
  • An amplification reaction product refers to the mixture of nucleic acids produced by completing an amplification reaction with a capture reaction product.
  • Figure 2 shows a schematic of a protocol wherein the probe circularizes to capture a region of the target nucleic acid and is then amplified by universal primers in preparation for sequencing.
  • a “homologous probe sequence” is a portion of a probe provided by the invention that specifically hybridizes to a target sequence present in the genome of a hepatitis C virus.
  • the terms “homologous probe sequence,” “probe arm,” “homologous probe arm,” “homer,” and “probe homology region” each refer to homologous probe sequences that may specifically hybridize to target genomic sequences, and are used interchangeably herein.
  • “Target sequence” refers to a nucleic acid sequence on a single strand of nucleic acid in the genome of an organism of interest. In some embodiments, the homologous probe sequences in the probes are the probe pairs listed in tables 1-4.
  • hybridizes refers to sequence-specific interactions between nucleic acids by Watson-Crick base-pairing (A with T or U and G with C). "Specifically hybridizes” means a nucleic acid hybridizes to a target sequence with a T m of not more than 14 °C below that of a perfect complement to the target sequence.
  • a bridge nucleic acid may be employed, wherein at least a first portion of the bridge nucleic acid is capable of hybridizing to the capture probe, and at least a second portion of the bridge nucleic acid (which may overlap with the first portion) is capable of simultaneously or sequentially hybridizing to the target nucleic acid, thereby enhancing the efficiency of ligation of the capture probe to the target.
  • a probe specifically hybridizes when: a) both homologous probe sequences A and C in the probe hybridize to their respective target sequence with at least 60, 65, 70, 75, 80, 85, 90, 95, or 100% correct pairing across the entire length of the homologous probe sequence; b) the 3'-most homologous probe sequence (also referred to herein as "C") hybridizes with 100% correct pairing in the 8, 7, 6, 5, 4, 3, or 2 bases at the 3' end of probe sequence C; and c) the 5'-most homologous probe sequence (also referred to herein as "A”) hybridizes the first 8, 7, 6, 5, 4, 3, or 2 bases of the 5' end of probe sequence A.
  • both homologous probe sequences A and C in the probe hybridize to their respective target sequence with at least 60, 65, 70, 75, 80, 85, 90, 95, or 100% correct pairing across the entire length of the homologous probe sequence
  • the 3'-most homologous probe sequence also referred to herein as "C” hybridizes with 100% correct pairing in
  • a probe specifically hybridizes when: a) both homologous probe sequences A and C in the probe hybridize to their respective target sequence with at least 80% correct pairing across the entire length of the homologous probe sequence, b) homologous probe sequence C hybridizes with 100% correct pairing of the first 6 bases of the 3' end of C; and c) homologous probe sequence A hybridizes with 100% correct pairing of the first 6 bases of the 5' end of A.
  • a probe specifically hybridizes when: a) both homologous probe sequences A and C in the probe hybridize to their respective target sequences with a melting temperature that is within some range (eg, 10 degrees Celsius given the hybridization buffer) of the perfect match Tm, b) both homologous probe sequences hybridize with 100% correct pairing over the 6 bases at the external ends.
  • Homology between two sequences may be determined by any means known in the art, including pairwise alignment, dot-matrix, and dynamic programming, and in particular embodiments by FASTA (Lipman and Pearson, Science, 227: 1435-41 (1985) and Lipman and Pearson, PNAS, 85: 2444-48 (1998)), BLAST (McGinnis & Madden, Nucleic Acids Res., 32:W20-W25 (2004) (current BLAST reference, describing, inter alia, MegaBlast); Zhang et al. , J. Comput.
  • FASTA Lipman and Pearson, Science, 227: 1435-41 (1985) and Lipman and Pearson, PNAS, 85: 2444-48 (1998)
  • BLAST McGinnis & Madden, Nucleic Acids Res., 32:W20-W25 (2004) (current BLAST reference, describing, inter alia, MegaBlast); Zhang et al. , J. Comput.
  • the methods provided by the invention comprise screening candidate sets of sequences by MegaBLAST against one or more annotated genomes.
  • a sequence “specifically hybridizes” when it hybridizes to a target sequence under stringent hybridization conditions.
  • Stringent hybridization conditions refers to hybridizing nucleic acids in 6xSSC and 1 % SDS at 65 °C, with a first wash for 10 minutes at about 42 °C with about 20% (v/v) formamide in O.lxSSC, and a subsequent wash with 0.2xSSC and 0.1 % SDS at 65 °C.
  • alternate hybridization conditions can include different hybridization and/or wash temperatures of about 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 66, 67, 68, 69, or 70 °C or other hybridization conditions as disclosed in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition (2001 ), which is incorporated herein by reference.
  • the hybridization temperature is greater than 60 °C, e.g., 60-65 °C.
  • homologous probe arms must be chosen to match the hybridization conditions, and vice-versa. If homologous probe arms are to be used at a less stringent hybridization condition (e.g. , a lower hybridization temperature or higher salt
  • nucleotides can be removed from the backbone-adjacent ends of the probe arms until the melting temperature of the probe arms is suitable for the
  • the melting temperature of the probe arm can be computed using any software known to those skilled in the art, such as Melting 5.0
  • nucleotides may be added to the backbone-adjacent ends of the probe arms such that the nucleotides match as many desired targets of the probe as possible.
  • the backbone-distal ends of the probe arms cannot be easily modified to change the melting temperatures of the arms as the nucleotide composition of those ends (typically the terminal two nucleotides) play a significant role in the efficiency of the polymerase initiation and the ligation reactions.
  • An “organism” is any biologic with a genome, including viruses, bacteria, archaea, and eukaryotes including plantae, fungi, protists, and animals.
  • Regular Interest refers to the sequence between the nearest termini of the two target sequences of the homologous probe sequences in a probe.
  • Homologous probe sequences A and C in a probe provided by the invention can readily be adapted for use as a pair of conventional primer pairs for use in a polymerase chain reaction (PCR) to specifically amplify a region of interest from a viral sequence.
  • "Conventional primer pairs” refers to a pair of linear nucleic acid primers each member of which comprises sequences corresponding to one of the two homologous probe sequences in a probe provided by the invention, which are capable of exponential amplification of a region of interest comprising at least two nucleotides. These conventional primer pairs are encompassed by and are a part of the present invention.
  • a convention primer pair comprises a first primer comprising the sequence of an extension arm of a circularizing capture probe provided by the invention— i.e. a 3' primer or "C" sequence” and a second primer comprising the reverse complement of a ligation arm of a circularizing capture probe provided by the invention— i.e. a 5' primer or "A" sequence.
  • the conventional primer pairs comprise a barcode sequence.
  • the conventional primer pairs comprise universal sequences, including, for example, sequences that hybridize to adaptamer primers.
  • the probes and conventional primer pairs provided by the invention may comprise the naturally occurring conventional nucleotides A, C, G, T, and U (in deoxyriobose and/or ribose forms) as well as modified nucleotides such as 2'O-Methyl- modified nucleotides (Dunlap et al, Biochemistry. 10(13):2581-7 (1971 )), artificial base pairs such as IsodC or IsodG, or abasic furans (such as dSpacer) (Chakravorty, et al. Methods Mol Biol.
  • the 5' or 3' homologous probe sequences of a probe provided by the invention comprise, at their respective termini, a photocleavable blocking group, such as PC-biotin.
  • a probe provided by the invention comprises a photocleavable blocking group at its 5' terminus to block ligation until photoactivation.
  • a probe provided by the invention comprises at its 3' terminus a photocleavable blocking group to block polymerase-dependent extension or n-mer oligonucleotide ligation until photoactivation.
  • the 5'-most nucleotide of a probe provided by the invention comprises an adenylated nucleotide to improve ligation and/or hybridization efficiency. See, e.g., Hogrefe et al., J Biol. Chem. 265 (10): 5561-5566, (1990).
  • the 5' end of the 5' homologous probe region (e.g., the ligation arm) comprises at least one LNA and in still more particular embodiments, the 5' terminal nucleotide is a LNA.
  • the probe molecule is capped with a phosphate group at the 5' end to improve the ligation efficiency.
  • Homologous probe arms may be chosen to capture regions that identify particular HCV genotypes or sub-genotypes.
  • the set of 27 pairs of probe arms listed in tables 1-4 were chosen to distinguish among genotypes 1 a, 1 b, 1g, 2a, 2b, 3, 6a, 6m, 6k, 6n, 6j, 6i, and 6f. These probe arms were designed against regions that are relatively conserved across 661 instances of the above genotypes but that surround capture regions that are relatively variable.
  • AF169004 3749 3919 ACCAGATACAGGTCGACCGCT GGATGAAGTCTATGGACTTAGC
  • EU155283 3754 3900 AGTAAGAGATAGGCCGGGGCG TGGAGAAGAGTTGTCTGTGAAC
  • EU155315 3607 3762 CCTGGTCTACATTGGTGTACATCT GAAGAGCCCTTCAAGTAGGAG
  • EU256069 3550 3712 GTACATCTGGATGACAGGACCCT CTTTCAAATAAGAGATAGGCCGG
  • FJ435090 3744 3911 GTCACTAGATAGAGATCTGATGCGC ACGAAATCAAGTGCTTTCGC
  • EU155331 6341 6517 ACTCCCTTATACCCACGTTGGC CCATAGCGCCCTAGAATAGTT
  • AF165052 8360 8535 GCTCTGTGAGCGACTTTATGGC AGATAACGACTAGGTCGTCTC
  • EU256064 8330 8494 ACATAAAGCCGCTCTGTGAGCG GATAACGACAAGGTCGTCTCC
  • BBBBBBBB represents a unique barcoding sequence and TxC and AxC indicate a phosphorothioate bond between the T and C or A and C.
  • Homologous probe arms may also be chosen to capture drug resistance mutations. These sets of probes were chosen to capture one or more drug resistance mutations across a set of 661 full or partial HCV genomes that are publically available. See e.gJ/hcv.lanl.gov; HCV sequence database: Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos HCV Sequence Database. Bioinformatics(2005), 21(3):379-84. The probe selection process attempted to select three probes that would "work” against every drug resistance mutation listed in Table 5 in each of the 661 genomes. The software considers a probe to work in a strain if it is expected to capture with at least 10% of its maximum efficiency. Table 2 lists the 162 probes designed against drug resistance mutations in the NS3 protease gene. Table 3 lists the 119 probes designed against the NS5a gene. Table 4 lists the 129 probes designed against the NS5b polymerase gene.
  • the probes provided by the invention include a probe backbone sequence between the first and second homologous probe sequences.
  • the backbone sequence can be at least 15, 20, 25, 30, 35, 40, 45, 50, 70, 90, 100, 12, 140, 150, 160, 180, 200, 400 bases, or more.
  • the backbone sequence may include a detectable moiety.
  • the detectable moiety is a probe-specific sequence, such as a barcode for identification of a specific probe or set of probes.
  • the backbone sequence comprises one or more primer- binding sites.
  • the backbone includes two primer- binding sites.
  • Each backbone primer-binding site may comprise one or more universal sequences that, for example, can be used to amplify all circularized probes in a mixture.
  • the backbone sequence comprises one or more non Watson- Crick nucleotides.
  • the backbone comprises one or more 2'0Methyl nucleotide residues, artificial base pairs such as IsodC or IsodG, or abasic furans (such as dSpacer), or 2'OMethyl, abasic furans, or LNA nucleotides, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more LNAs or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%
  • 2'OMethyl, abasic furans, or LNA nucleotides to confer greater reactivity or inertness in the hybridization reaction, provide resistance to enzymatic activities such as
  • polymerase-mediated strand displacement or nuclease cleavage to serve as inhibitors of spurious amplification events, or to act as target sites for trans-acting nucleic acid oligonucleotides such as PCR primers or biotinylated capture probes.
  • the backbone sequence B comprises a cleavage site.
  • the cleavage site is a restriction endonuclease recognition site.
  • the backbone sequence B comprises one or more detectable moieties.
  • the one or more detectable moieties are each independently selected from a barcode sequence and a primer-binding sequence.
  • the backbone contains non-Watson-Crick nucleotides, including, for example, abasic furan moieties, and the like.
  • the backbone comprises the sequence
  • the backbone comprises the sequence GTTGGAGGCTCATCGTTCCTATATTCCACACCACTTATTATTACAGATGTTATGCTCG CAGGTC.
  • barcode is used to refer to a nucleotide sequence that uniquely identifies a molecule or class of related molecules.
  • Suitable barcode sequences that may be used in the probes of the invention may include, for example, sequences corresponding to customized or prefabricated nucleic acid arrays, such as n-mer arrays as described in U.S. Patent No. 5,445,934 to Fodor er a/, and U.S. Patent No.
  • the n-mer barcode may be at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 nucleotides, e.g., from 18 to 20, 21 , 22, 23, 24, or 25 nucleotides.
  • the n-mer barcode is from 6 to 8 nucleotides.
  • the n-mer barcode is from 10 to 12 nucleotides.
  • the barcodes include sequences that have been designed to require greater than 1 , 2, 3, 4 or 5 sequencing errors to allow this barcode to be inadvertently read as another in error.
  • the probe does not contain a barcode, while a primer that is used to amplify a circularized probe contains a barcode.
  • barcode sequences for each barcode size K, 4 K random barcodes may be generated from the four DNA nucleotides, A, T, G, C, using a Perl script.
  • This set of barcodes represents the total number of unique sequence combinations possible for a sequence of K length, using 4 nucleotide variations. Barcodes for which one nucleotide comprises 100% of the length, e.g., TTTTTT, are then optionally removed using a pattern-matching Perl script. Further filtering steps may include removal of barcodes which contain runs of nucleotides of >3, e.g., TGGGGT, or runs interrupted by only one nucleotide, for instance, GGGTGG.
  • Barcodes containing palindromes or inverted repeats with a propensity to form secondary structure through self-hybridization may be filtered using a Perl script designed to identify such self-complementarity.
  • a set of candidate barcodes may be further filtered such that every barcode contains at least some number of base differences compared to any other barcode.
  • barcodes may be selected to be an edit distance of two nucleotides apart (i.e., differing in sequence by two nucleotides) to ensure that a single sequencing error does not cause barcode mis-identification.
  • Selection of barcodes that may be utilized in a mixture of probes used to test a sample from a patient may involve selecting a combination of barcodes that will provide >5% and not more than 50% representation of a particular nucleotide at each position in the barcode sequence within the pool. This is achieved by random addition and removal of barcodes to a pooled set until the conditions specified are met using a Perl script. Barcodes for which the reverse complement sequence is also present within the barcode pool may also be eliminated.
  • Suitable barcode sequences include such barcode sequences as set forth in the table below, which illustrates exemplary 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9- mer, and 10-mer barcode sequences.
  • Sequences indicated as “1 nucleotide distance” nmers in Table 3 are illustrative sequences that have a sequence distance of at least 1 from each other, where “distance” refers to the minimum number of sequencing differences between each of the sequences of the same category.
  • “Two nucleotide distance” sequences have a "distance" from each other of at least 2 nucleotides.
  • barcodes used in the probes provided by the invention correspond to those on the Tag3 or Tag4 barcode arrays by AFFYMETRIXTM. Further discussion of barcode systems can be found in Frank, BMC Bioinformatics, 10:362 (2009; 13 pages), Pierce er a/. , Nature Methods, 3:601-03 (2006) (including web supplements), and Pierce et a/., Nature Protocols, 2:2958-74 (2007).
  • the barcode is sample-specific, e.g. , comprises one or more patient specific barcodes.
  • more than one barcode will be assigned per patient sample, allowing replicate samples for each patient to be performed within the same sequencing reaction.
  • the barcode may be temporal, e.g., a barcode that specifies a particular period of time.
  • a temporal barcode it is possible to detect carry-over or contamination on an assay instrument, such as a sequencing instrument, between runs on different days.
  • sample and/or temporal barcodes may be used to automatically detect cross-contamination between samples and/or days and, for example, instruct an instrument operator to clean and/or decontaminate a sample handling system, such as a sequencing instrument.
  • a barcode sequence in the backbone is located between a universal primer binding site and a probe arm such that the barcode sequence is amplified by the universal primer.
  • universal primer binding sequences in a backbone sequence serve as a hybridizing template for longer "adaptamer” primers.
  • adaptamer primer is a primer that hybridizes to universal primer sequences in a capture reaction product to facilitate amplification of the capture reaction product and further comprise a sample-specific barcode sequence, e.g., sequence 5' to the universal primer hybridizing region of the adaptamer primer.
  • Adaptamer primers can be used, for example, to incorporate sample-specific barcodes on amplification reaction products to allow further multiplexing of samples after completing a capture reaction and an amplification reaction.
  • the addition of sample-specific barcodes allows multiple capture and/or amplification reaction products to be pooled before detection by, for example, sequencing.
  • the adaptamer primers further include universal sequences that hybridize to a sequencing primer.
  • the detectable moiety may be associated with the backbone sequence. It may be bound to the polynucleotide sequence, as in the case of direct labels, such as fluorescent (e.g., quantum dots, small molecules, or fluorescent proteins), chemical or protein-based labels. Alternatively, the detectable moiety may be incorporated within the polynucleotide sequence, as in the case of nucleic acid labels, such as modified nucleotides or probe-specific sequences, such as barcodes. Quantum dots are known in the art and are described in, e.g., International Publication No. WO 03/003015.
  • the backbone may also contain a "random" sequence, typically written as a sequence of N's. Such random sequence indicates that any of the four nucleotides A, T, C, or G will be incorporated at that position in the synthesized probe molecule. Within a population of probe molecules, one expects to see all or many of the 4 ⁇ ⁇ possible sequences for the string of Ns. For example, a probe with the backbone
  • GTTGGAGGCTCATCGTTCCTATATTCCACACCACTTATTATTANNNNNNCAGATGTTA TGCTCGCAGGTC could actually by one of 4 6 molecules. Including such random sequences, also known as "dogtags," in the probe molecule allows one skilled in the art to determine the most likely number of circularized probe molecules after amplification and sequencing by counting the number of unique dogtags seen. 2 Probe Mixtures
  • aspects of the invention provide one or more probes for multiplex analysis of test samples, including hepatitis virus detection and hepatitis C viral genotyping in a biological sample from a patient.
  • aspects of the invention encompass a composition comprising a plurality of probes, each comprising a nucleic acid sequence of the formula:
  • A is a probe arm sequence taken from column 2 of tables 1-4;
  • C is a corresponding probe arm sequence from column 3 of tables 1-4;
  • B is a backbone sequence.
  • each probe arm sequence in the group consisting of all sequences from tables 1-4 is contained in at least one probe in the plurality of probes.
  • Probes in a mixture may be selected such that the mixture comprises a subset of the full group of probes encompassed by the probe arm sequence pairs provided in tables 1-4, so as to detect a particular subset of hepatitis C genotypes or a particular subset of mutations.
  • Probes in a mixture will typically have similar bulk properties (such as,
  • homologous probe sequence length the homologous probe sequence T m , and length of the captured region of interest, and the lack of secondary structure
  • the T m of the homologous probe sequences in a mixture of probes will be within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 °C of each other, or in particular embodiments have the same T m .
  • the homologous probe sequences in a mixture of probes will all be within 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide in length of each other.
  • the length of the region of interest between the target sequences of probes in a mixture may vary over a range of values, such as from 2 to 20, 20 to 100, 20 to 200, 40 to 300, 100 to 300, 100 to 500, 80 to 500, or 100-180 nucleotides. In some embodiments, the length of the region of interest between the target sequences of probes in a mixture is from 100 to 489 nucleotides. In particular embodiments, the regions of interest are within 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides in length of each other. Barcode lengths may also vary, but are generally within 25, 20, 15, 10, or 5 nucleotides of each other. In particular embodiments, the barcodes are the same length.
  • mixtures provided by the invention comprise capture reaction products and amplification reaction products from different test samples, as further described below.
  • different capture reaction products and/or amplification reaction products can be combined and multiplexed before detection, i.e., for concurrent detection. This is accomplished using barcode sequences that identify the test samples.
  • capture reaction products from test sample A will include a sample A-specific barcode
  • capture reaction products from sample B will include a sample B-specific barcode.
  • all sequences in the sample A capture reaction products are identified by the presence of the sample A-specific barcode sequence.
  • the mixtures of the invention contain sample internal calibration nucleic acids (SICs).
  • SICs sample internal calibration nucleic acids
  • known quantities of one or more SICs are included in a mixture provided by the invention.
  • the SICs have a nucleotide composition characteristic of pathogenic DNA targets and are present in specific molar quantities that allow for reconstruction of a calibration curve for quality control, e.g., for the processing and sequencing steps for each individual test sample.
  • the SICs makes up approximately 10% (molar quantity) of nucleic acids in a mixture, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20% (molar) of nucleic acids in the mixture.
  • different SICs are present in different concentrations, for example, in a dilution series, over a 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 5000, 10000, 50000, or 100000 -fold concentration range from the most dilute to most concentrated SICs in 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 steps.
  • SICs are present in a sample (e.g., a mixture of probes and a test sample, a capture reaction, a capture reaction product, an amplification reaction, or an amplification reaction product) at concentrations of 5, 25, 100, and 250 copies/ml.
  • an organism count per unit volume e.g., copies/mL for liquid samples such as blood or urine
  • an organism count per unit volume can be estimated for each organism detected.
  • the concentration of SICs and probes directed to the SICs are adjusted empirically so that sequences of SICs detected in a capture reaction product and/or amplification reaction product make up about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, or 30% of sequences in the mixture.
  • SICs make up 10-20% of sequence reads.
  • the number of SICs sequence reads in a sequencing reaction is quantitatively evaluated to ensure that sample processing occurs within pre-defined parameters.
  • the pre- defined parameters include one or more of the following: reproducibility within two standard deviations relative to all samples sequenced during a particular run, empirically determined criteria for reliable sequencing data (e.g., base calling reliability, error scores, percentage composition of total sequencing reads for each probe per target organism), no greater than about 15% deviation of GC or AU-rich SICs within a sequencing run.
  • the SICs DNA in a sample will also comprise the same barcode(s) corresponding to unique samples, e.g., particular patient samples.
  • SICs may comprise a region of interest as defined above, where the region of interest is modified to further comprise a sequence heterologous to the region of interest.
  • the sequence heterologous to the region of interest in the SICs is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 contiguous bases, or more.
  • the SIC contains the nucleotide sequence
  • the probe mixture includes the probe with sequence 5- /5Phos/GTG GTA TGG CTG ATT ATG ATC TAG AGT GTT GGA GGC TCA TCG TTC CTA TAT TCC TGA CTC CTC ATT GAT GAT TAC AGA TGT TAT GCT CGC AGG TCG AGT TTG GAC AAA CCA CAA CTA GAA -3.
  • the mixtures of the invention contain sample nucleic acids.
  • the nucleic acids may be obtained from any test sample, such as a biological sample.
  • the nucleic acids obtained from the test sample may be of varying degrees of purity, such as at least 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99% of organic matter by weight.
  • the sample nucleic acids are extracted from a test sample.
  • Test samples may be from any source and include swabs or extracts of any surface, or biological samples, such as patient samples.
  • Patients may be of any age, including adults, adolescents, and infants.
  • Biological samples from a subject or patient may include blood, whole cells, tissues, or organs, or biopsies comprising tissues originating from any of the three primordial germ layers— ectoderm, mesoderm or endoderm.
  • Exemplary cell or tissue sources include skin, heart, skeletal muscle, smooth muscle, kidney, liver, lungs, bone, pancreas, central nervous tissue, peripheral nervous tissue, circulatory tissue, lymphoid tissue, intestine, spleen, thyroid, connective tissue, or gonad.
  • Test samples may be obtained and immediately assayed or, alternatively processed by mixing, chemical treatment, fixation/ preservation, freezing, or culturing.
  • Biological samples from a subject include blood, pleural fluid, milk, colostrums, lymph, serum, plasma, urine, cerebrospinal fluid, synovial fluid, saliva, semen, tears, and feces.
  • the biological sample is blood.
  • Other samples include swabs, washes, lavages, discharges, or aspirates (such as, nasal, oral, nasopharyngeal, oropharyngeal, esophagal, gastric, rectal, or vaginal, swabs, washes, ravages, discharges, or aspirates), and combinations thereof, including combinations with any of the preceding biopsy materials.
  • the invention provides a method for detecting the presence of one or more hepatitis C virus by contacting a sample suspected of containing at least one such virus with a mixture of probes of the invention, capturing a region of interest of the at least one virus (e.g., by polymerization and/or ligation) to form a circularized probe, and detecting the captured region of interest, thereby detecting the presence of the one or more hepatitis C viruses.
  • the captured region of interest may be amplified to form a plurality of amplicons (e.g., by PCR).
  • the sample is treated with nucleases to remove the linear nucleic acids after probe-circularizing capture of the region of interest.
  • the circularized probe is linearized, e.g., by nuclease treatment.
  • the circularized probe molecule is sequenced directly by any means known in the art, without amplification.
  • the circularized probe is contacted by an oligonucleotide that primes polymerase-mediated extension of the molecules to generate sequences complementary to that of the circularized probe, including from at least one to as many as 1 million or more concatemerized copies of the original circular probe.
  • the circularized probe molecule is enriched from the reaction solution by means of a secondary-capture oligonucleotide capture probe.
  • a secondary-capture oligonucleotide capture probe may comprise a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe.
  • the nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe may include 1 ,
  • the probe and/or captured region of interest is sequenced by any means known in the art, such as polymerase-dependent sequencing (including, dideoxy sequencing, pyrosequencing, and sequencing by synthesis) or ligase based sequencing (e.g., polony sequencing).
  • the sample is a biological sample.
  • the biological sample is from a mammal, such as a human.
  • the methods of detecting the presence of one or more hepatitis viruses further comprise the step of formatting the results to facilitate physician decision making by, for example, providing one or more graphical displays.
  • the invention provides a method of treating a subject suspected of being infected with a hepatitis C virus, comprising detecting at least one hepatitis C virus by the methods of the invention and administering a suitable therapeutic treatment based on the at least one hepatitis virus detected.
  • the invention also provides a method of treating a subject suspected of being infected with an HCV strain carrying a drug resistance mutation, comprising detecting at least one HCV drug resistant genotype by the methods of the invention and
  • HCV RNA may be directly contacted by probes or may be converted to DNA to be used with the probes disclosed in this invention.
  • Many techniques for converting RNA to DNA are available in the scientific literature. For example, random hexamer or octamer primers can be used with a reverse-transcriptase to generate first strand cDNA from the viral RNA. While simple, random priming also amplifies host (eg, human) RNA that may be present in a clinical sample, thus limiting the amount of viral cDNA produced by the reaction.
  • Figure 4 panels A and B depict cDNA generation approaches for embodiments of this invention.
  • Figure 5 shows the process from RNA through analysis.
  • a preferred embodiment of this invention uses a set of HCV-specific RT primers in an RT-PCR reaction to generate and amplify DNA from the viral RNA molecules. Similar to probes, good RT primers hybridize to relatively conserved portions of the HCV genome.
  • HCV genotypes 1 a and 1 b are the dominant strains.
  • Table 6 shows a set of RT primers that amplify the NS3, NS5A, and NS5B genes from genotypes 1 a and 1 b. These primers can be used with the Qiagen OneStep RT-PCR to produce cDNA from RNA that has been extracted from HCV blood samples.
  • a set of RT-PCR primers that target all known HCV genotypes may be used.
  • Table 7 lists a set of 75 15mers that achieve this goal for the NS3 gene.
  • This set of primer set works at a lower temperature (hybridization temperature at 30 °C ).
  • the invention provides methods of detecting the presence of one or more HCV strains in a test sample.
  • the methods comprise the step of contacting a mixture comprising probes described above with any of the test samples described above in a capture reaction, as defined above.
  • a mixture comprising probes is contacted with nucleic acids extracted from a test sample such as blood, along with a polymerase enzyme and nucleotide triphosphates (NTPs), and capturing at least one region of interest by polymerase-dependent extension of at least one homologous probe sequence in the mixture.
  • NTPs nucleotide triphosphates
  • the polymerase-dependent extension of a homologous probe sequence is followed by a ligation of the end of the extended (i.e., by the polymerase) homologous probe sequence to the end of the other homologous probe sequence to produce a circularized probe containing a region of interest from the genome of an HCV strain.
  • the ligation reaction occurs while the target arm is hybridized to the target.
  • the target arm is dissociated from the target and ligated in solution under reaction conditions favoring self-ligation over trans-ligation to other probe molecules, for example a dilute ligation solution.
  • Figure 2 illustrates one particular embodiment of a method provided by the invention. Briefly, hybridization of a probe to the target sequences in the organism of interest is followed by polymerase-mediated, target-sequence-directed addition of nucleotides to the 3' homologous probe sequence, terminating due to obstruction at the 5' homologous probe sequence of the probe. A ligation reaction joins the terminal 3' nucleotide to the 5' nucleotide of arm.
  • the sample may be treated with exonuclease to digest single stranded linear DNA.
  • Primers complementary to the probe backbone may amplify the MIP into dsDNA for sequencing.
  • amplification primers at this stage may contain sample-specific nucleotide barcode sequences, e.g., they may be adaptamer primers.
  • a unique primenbarcode molecule sequence therefore may identify each test sample. For example, a panel of 100 probes is contacted with 50 individual test samples. The homologous probe sequences detected in a sequence read identifies a strain of hepatitis C or a drug resistance genotype of a strain of HCV. Each test sample amplification reaction is done with one unique probe set.
  • Each barcode within the amplification primer can be used to act as an identifier for a patient, e.g., contains a barcode. Therefore 50 pairs of amplification primers (one for each amplification reaction product) and one panel of probes (e.g., probes for hepatitis A, B, and C distinction, for HCV genotyping, or both) are required for a 50-sample multiplex assay.
  • Polymerases for use in the methods provided by the invention include Taq polymerase (Lawyer et al., J. Biol. Chem., 264:6427-6437 (1989); Genbank
  • accession: P19821 including the 5'->3' nuclease deficient "Stoffel” fragment described in Lawyer et al., PCR Meth. Appl., 2:275-287 (1993)), PHUSIONTM high fidelity recombinant polymerase (NEB), and Pyrococcus furiosus (Pfu) polymerase (see, e.g., U.S. Patent No. 5,545,552), as well as polymerases comprising a helix-hairpin-helix domain, such as TopoTaq and PfuC2 (Pavlov et al., PNAS, 99:13510-15 (2002)).
  • the polymerase is 5'->3' nuclease deficient, such as the Stoffel fragment of Taq polymerase, which further lacks 3'- 5' proofreading activity.
  • Polymerases lacking 5'- 3' exonuclease activity may be generated by means known in the art, for example, based on methods of screening or rational design.
  • polymerase variants can be designed based on sequence alignments of one or more polymerases to the Stoffel fragment of Taq and/or by "threading" a sequence through a solved polymerase structure (e.g., MMDB IDs 56530, 81884 and 81885).
  • a polymerase for use in the methods of the invention is a non-displacing polymerase, such as Pfu, T4 DNA polymerase, or T7 DNA polymerase.
  • a polymerase for use in the methods provided by the invention is a polymerase suitable for isothermal amplification and capture and/or amplification reactions are performed isothermally, e.g., by controlling metal ion concentration and/or using particular polymerases and/or additional enzymes, such as helicases or nicking enzymes (such as primer generation RCA and EXPAR). See, e.g., U.S. Patent No. 6,566,103, Murakami et al., Nucl. Acid.
  • Polymerases foruse in isothermal amplification include, for example, Bst, Bsu andphi29 DNA polymerases, and E.coli DNA polymerase I.
  • a mixture of probes is contacted with nucleic acids extracted from a test sample, a ligase enzyme, and a pool of n-mer oligonucleotides in a capture reaction, as defined above.
  • the n-mer is contacted with nucleic acids extracted from a test sample, a ligase enzyme, and a pool of n-mer oligonucleotides in a capture reaction, as defined above.
  • the n-mer is contacted with nucleic acids extracted from a test sample, a ligase enzyme, and a pool of n-mer oligonucleotides in a capture reaction, as defined above.
  • the n-mer is contacted with nucleic acids extracted from a test sample, a ligase enzyme, and a pool of n-mer oligonucleotides in a capture reaction, as defined above.
  • the n-mer is contacted with nucleic acids extracted from a test sample, a ligas
  • oligonucleotides are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 or 25 nucleotides long. In more particular embodiments, they are random hexamers. In other embodiments, they are polynucleotides, the length of the region of interest between the first and second target sequences that hybridize to the homologous probe sequence. In some embodiments, the n-mer oligonucleotide contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 locked nucleic acids (LNAs) or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% LNAs.
  • LNAs locked nucleic acids
  • the ligase enzyme ligates the n-mer oligonucleotides with the probes provided by the invention to produce a circularized probe containing a region of interest from HCV.
  • Primers complementary to the probe backbone amplify the probe into dsDNA for sequencing.
  • amplification primers are adaptamer primers and contain sample-identifying barcode sequences. A unique barcode sequence therefore identifies each sample in a multiplex.
  • Each strain of HCV is identified by the unique combination of homologous probe sequences and ligated n- mer in a sequence read.
  • Ligases for use in the methods of the invention include T4, T7, and thermostable ligases, such a Taq ligase (as disclosed in Takahashi er a/., J. Biol. Chem., 259:10041 - 47 (1984), and international publication WO 91/17239), and AMPLIGASETM
  • mixtures comprising pairs of conventional PCR primers (conventional primer pairs) provided by the invention are contacted with sample nucleic acids to amplify a region of interest between two target regions in HCV.
  • a limited number of amplification steps are performed.
  • fewer than 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cycles of amplification are performed.
  • the mixture of conventional primer pairs are contacted with nucleic acids extracted from a test sample, a
  • primers binding to universal probe recognition sequence e.g., a barcode
  • conventional primer pairs can be used in a variety of additional methods.
  • conventional primer pairs may be contacted with a sample nucleic acid suspected of containing at least one target nucleic acid.
  • PCR may be used to amplify the region of interest directly from a sample nucleic acid.
  • the conventional primer pairs may be used to amplify capture reaction products, e.g., one or more circularized probes.
  • a sample nucleic acid suspected of containing a region of interest is amplified using a conventional primer pair and then contacted with a probe provided by the invention for circularizing capture.
  • conventional primer pairs are contacted with a sample nucleic acid and modified nucleotides, such as biotinylated nucleotides.
  • modified nucleotides such as biotinylated nucleotides
  • the resulting capture or amplification reaction products can then be isolated by affinity capture, for example, with steptavidin substrates, for subsequent processing, e.g., circularizing capture with the probes provided by the invention.
  • a single conventional primer may be used for linear amplification of a region of interest in a sample nucleic acid in, and then contacted with a probe provided by the invention for circularizing capture.
  • a single conventional primer containing a 5' biotin moiety may be used to amplify a target sequence and then be enriched from the sample using streptavidin capture for sequencing by, for example, direct sequencing using either specific conventional primer pairs provided by the invention, or by random hexamer priming, or may be used for circularizing capture using probes provided by the invention
  • methods that comprise a capture reaction further comprise the step of contacting the capture reaction product with one or more
  • exonuclease includes at least one of exo I, exo III, exo VII, and exo V.
  • the exonuclease is up to a 100:1 , 50:1 , 25:1 , 10:1 , 5:1 , 2:1 , 1 : 1 , 1 :2, 1 :5, 1 :10, 1 :25, 1 :50, or 1 : 100 (unit to unit) mixture of exonuclease I and
  • the methods of the invention further comprise the step of amplifying capture reaction products in an amplification reaction.
  • amplifying nucleic acids include the polymerase chain reaction (see, e.g. , U.S. Patent Nos. 4,683,195 and 4,683,202 and McPherson and Moller, PCR (the baSICs), Taylor & Francis; 2 edition (March 30, 2006)), OLA (oligonucleotide ligation amplification) (see, e.g., U.S. Patent Nos. 5,185,243, 5,679,524, and
  • amplification is linear amplification such as, RCA.
  • capture reaction products e.g., circularized probes
  • the RCA reaction may comprise contacting a sample with modified nucleotides, such as biotinylated nucleotides, LNA nucleotides or artificial base pairs such as IsodC or IsodG, or abasic furans (such as dSpacer), to facilitate affinity enrichment and purification.
  • the amplification reaction products comprising linear repeating ssDNA can be contacted with a conventional primer provided by the invention to produce short extensions of double stranded DNA with a length 2, 3, 4, 5 ,6, 7,10,15, 20, 30, 40, 50, 75, 100, 500 nucleotides.
  • the length of extension may be controlled by time of extension step at the optimum temperature of elongation for this polymerase, e.g., 5, 10, 15, 20, 40, 60 seconds, at temperatures including 37, 42, 45, 68, 72, 74 °C.
  • the length of extension is controlled by mixing of nucleotide analogues that prevented further elongation into the reaction, such as dideoxyCytosine, or nucleotides with a 3' modification such as biotin, or a carbon spacer terminated with an amino group.
  • nucleotide analogues that prevented further elongation into the reaction, such as dideoxyCytosine, or nucleotides with a 3' modification such as biotin, or a carbon spacer terminated with an amino group.
  • a primer is contacted with a linear repeating ssDNA RCA amplification reaction product and extended by a polymerase for a single cycle of PCR, to generate a short single stranded DNA containing the complementary sequence to the repeating unit of the RCA product.
  • the primer contacted with a linear repeating ssDNA RCA amplification reaction product produces a dsDNA region comprising a restriction enzyme cleavage site. Accordingly, in certain embodiments, when the primer hybridizes to the linear repeating ssDNA RCA amplification reaction product to form a double-stranded DNA region, the amplification reaction product is contacted with the restriction enzyme to produce shorter fragments.
  • the amplification reaction uses adaptamer primers.
  • the amplification reaction uses sample-specific primers, that is, primers that hybridize to sequences present in the probe that identify the sample.
  • sample-specific primers that is, primers that hybridize to sequences present in the probe that identify the sample.
  • a low number of amplification cycles are used to avoid amplification artifacts, e.g., fewer than 25, 20, 15, 10, 9, 8, 7, 6, or 5 cycles.
  • the methods provided by the invention may comprise the step of contacting sample nucleic acids, capture reaction products or amplification reaction products with a secondary-capture oligonucleotide capture probe which comprises a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence, which is able to hybridize to the sample nucleic acids, capture reaction products, or amplification reaction products.
  • a secondary-capture oligonucleotide capture probe which comprises a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence, which is able to hybridize to the sample nucleic acids, capture reaction products, or amplification reaction products.
  • a secondary-capture oligonucleotide capture probe which comprises a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence, which is able to hybridize to the sample nucleic acids, capture reaction products, or amplification reaction products.
  • a biotinylated probe may be extended on sample nucleic acids, capture reaction products or amplification reaction products using thermophilic or mesophilic polymerases.
  • the method comprises contacting a capture reaction product with a biotinylated oligonucleotide for enrichment of specific capture reaction products using the biotin:streptavidin interaction.
  • Sequences captured by the methods of the invention can be detected by any means, including, for example, array hybridization or direct sequencing. In some embodiments, captured sequences may be detected by sequencing without
  • the sequencing methods rely on the specificity of either a DNA polymerase or DNA ligase and include, e.g., pyrosequencing, base extension sequencing (single base stepwise extensions), multi-base sequencing by synthesis (including, e.g. , sequencing with terminally-labeled nucleotides) and wobble sequencing, which is ligation-based.
  • sequencing technology used in the methods provided by the invention include Sanger sequencing, microelectrophoretic sequencing, nanopore sequencing, sequencing by hybridization (e.g. , array-based sequencing), realtime observation of single molecules, and cyclic-array sequencing, including
  • pyrosequencing e.g. , 454 SEQUENCING ® , see, e.g., Margulies et al. , Nature, 437: 376-380 (2005)
  • ILLUMINA ® or SOLEXA ® sequencing ⁇ see, e.g., Turcatti et al., Nucleic Acids Res., 36, e25 (2008), see also U.S. Patent Nos. 7,598,035, 7,282,370, 7,232,656, and 7,115,400), polony sequencing (e.g. , SOLiDTM, see Shendure et al. 2005), and sequencing by synthesis (e.g. , HELICOS ® , see, e.g., Harris et al., Science, 320:106-109 (2008)).
  • the capture probes contain sequences that facilitate processing for sequencing by a certain sequencing technology, such as sequences that can serve as anchor sites for sequencing by synthesis, primer sites for sequencing reaction initiation, or restriction enzyme sites that allow cleavage for improved ligation of oligonucleotide adaptors for sequencing of the particular amplicon.
  • sequences that facilitate processing for sequencing by a certain sequencing technology such as sequences that can serve as anchor sites for sequencing by synthesis, primer sites for sequencing reaction initiation, or restriction enzyme sites that allow cleavage for improved ligation of oligonucleotide adaptors for sequencing of the particular amplicon.
  • circularized capture probes are contacted by oligonucleotides which prime polymerase-mediated extension of the capture probes to generate sequences complementary to that of the circularized probe, including from at least one to one million or more concatemerized copies of the original circular probe.
  • homologous probe sequences may be used in the probes provided by the invention, as well as conventional primer pairs.
  • the homologous probe sequences will be about 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.
  • the region of interest between the target sequences of a probe or conventional primer pair is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 bases.
  • the probes provided by the invention may be circularized by polymerase-dependent synthesis and ligation, or by ligation of n-mer oligonucleotides of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 5, 20, 25, 30, 35, 40, 45, or 50 bases.
  • the region of interest is about 7 bases and homologous probe sequences are 10 or 12 bases.
  • a 7-mer oligonucleotide comprising a locked nucleic acid is ligated to a probe provided by the invention, and in still more particular embodiments, the 7-mer oligonucleotide comprises at least 1 , 2, 3, 4, 5, 6, or 7 locked nucleic acids (LNAs).
  • capture or amplification reaction products may be sequenced by emulsion droplet sequencing by synthesis as disclosed in, for example, Binladen er a/, PLoS One. 2(2):e197 (2007).
  • capture products may be amplified by RCA to generate higher copy numbers of capture product within a single DNA molecule in order to facilitate emulsion of captured DNA for emulsion PCR and sequencing by synthesis. See, e.g., Drmanac et al, Science 327(5961 ):78-81 (2010).
  • capture reaction products and/or amplification reaction products containing different samples are combined before detection.
  • capture and/or amplification reaction products are
  • combinatorially pooled before detection e.g., an MxN array of individual capture reaction products and/or amplification reaction products are pooled by row and column, and the pools are detected. Results from row and column pools can then be
  • capture reaction products and/or amplification reaction products contain identifying barcode sequences.
  • amplification primers contain sample-specific barcode sequences. Accordingly, the sample source of sequences contained in pools of capture reaction products and/or amplification reaction products are identified by their barcode sequences.
  • the methods provided by the invention may also include directly detecting a particular nucleic acid in a capture reaction product or amplification reaction product, such as a particular target amplicon or set of amplicons.
  • the mixtures of the invention comprise specialized probe sets including TAQMANTM, which uses a hydrolyzable probe containing detectable reporter and quencher moieties, which are released by a DNA polymerase with 5'- 3' exonuclease activity (U.S. Pat. No. 5,538,848); molecular beacon, which uses a hairpin probe with reporter and quenching moieties at opposite termini (U.S. Patent No. 5,925,517);
  • FRET fluorescence resonance energy transfer
  • the methods of the invention comprise using sample internal calibration nucleic acid (SICs) to estimate the concentration of a hepatitis strain in a test sample. This is done by calibrating the frequency of a sequence from a hepatitis strain to the known concentration of the SICs to provide an estimated concentration of the viral strain in the test sample.
  • the estimated concentration of the viral strain is compared to a database of reference concentrations of hepatitis strains associated with a disease state and/or likely clinical diagnoses.
  • the methods of the invention further comprise steps of formatting results to inform physician decision making.
  • Results refers to the outcome of detecting a target organism and includes, e.g., binary (e.g., +/-) detection as well as estimates of concentration, and may be based on, inter alia the result of sequencing a capture reaction product or amplification reaction product.
  • the formatting comprises presenting an estimate of the concentration of an organism in a test sample, optionally including statistical confidence intervals.
  • the formatting further comprises color-coding of the results.
  • the formatting includes recommendations for therapeutic intervention, including, for example, hospitalization, probiotic treatment, antibiotic treatments, and chemotherapy.
  • the formatting comprises one or more of the following: references to peer-reviewed medical literature and database statistics of empirically defined sample results.
  • Phusion polymerase is used to copy the reverse- complement of the target sequence into the probe and Ampligase ligase is used to circularize the resulting molecule as shown in Protocol 1 .
  • the resulting circular molecules can be amplified using adapter-primers shown in Table 8 to prepare the material for sequencing on either the Ion Torrent or lllumina platforms.
  • the probes are applied directly to extracted RNA without generating a cDNA intermediate.
  • This embodiment requires a DNA polymerase capable of using an RNA template (eg a standard reverse-transcriptase such as Tth,
  • Figure 5 shows the distribution of probes from tables 1-4 across a reference HCV genome.
  • the probes target 661 HCV strains from many genotypes.
  • the number of probes in each region indicates the sequence diversity in that region.
  • Conversion of raw sequence data may occur in three stages, namely (1) the processing of raw instrument data and conversion into aligned sequencing reads, (2) statistical interpretation of read data and (3) providing output and storage in archives.
  • statistical analysis and interpretation determine the most likely strains or substrains present in the sample given the sequencing data.
  • each sequencing read is first compared to the set of probe arms used in the capture reaction using an algorithm similar to Needleman- Wunsch but with no terminal gap penalty in the probe arm.
  • the software retains the probe arm with the best score. Having identified the probe arm and therefore
  • the software then compares the sequencing read against all expected reads for that probe, where expected reads were generated by an in-silico application of the probe set to a set of full or partial HCV genomes. All matches of a probe to a genome that meet some minimum criteria are included in the set of expected reads. Having compared all reads to all expected reads, the software picks the most likely strain or strains present in the sample based on the alignment scores, a model of mutation probabilities, and a user-provided prior probability on the number of strains to expect.
  • the methods of analysis determine the relative distance
  • the hidden variables in the model are the proportions or abundances of the strains and the assignments of sequencing reads to expected reads (where each observed read is assigned to a single expected read).
  • a variety of methods including Expectation- Maximization, Gibbs Sampling, and Metropolis-Hastings, may be used to find the values of these hidden variables, which maximize the probability of the data given the hidden variables and the priors on the hidden variables.
  • the software compares each read against both probe arms for each probe.
  • the software performs two alignments for each read-probe pair, first aligning the first probe arm with no terminal gap penalty for the probe arm and then aligning the other probe arm with no initial gap penalty for that probe arm.
  • the section of the read between the two probe arm alignments is the copied part of the target region that the probe captured from the target nucleic acid.
  • the software analyzes subsets of the data, where each subset contains only the capture regions of sequencing reads that overlap a mutation of interest.
  • Tools well known in the art such as
  • FreeBayes, SAMTools, and ShoRAH can be used to estimate the frequency of each allele based on the sequencing data.
  • Output of results can occur in parallel (1 ) to company server, (2) to xml and HL7 formats, e.g., for deposit in hospital system, in an electronic medical record (EMR) system, or in other HL7 or xml capable storage systems, for use in existing health record frameworks, and/or (3) to physician-friendly graphical and text formats, e.g., graphs, tables, summary text and possible annotated, web formats linking to reference information.
  • Output formats are arbitrary, e.g., simple text, spreadsheet data, binary data objects, encrypted and/or compressed files.
  • a complete record may involve all or some of these linked to a diagnostic test via unique identifiers. They may be assembled into a coherent object or may be accessible via a search for the unique identifier.
  • Protocols The following protocols were used in the examples described below and can be used by the skilled artisan when practicing the methods provided by the invention or using the probes, mixtures, and compositions provided by the invention. Variations on these protocols will be readily apparent to the skilled artisan.
  • Protocol 1 MIP capture, HCV cDNA target capture
  • thermocycler When the thermocycler reaches the 60° hold (approximately 26 minutes), add 2 ⁇ _ of enzyme mix to each sample and then advance the
  • thermocycler to the next step (60° for 10 min).
  • thermocycler When the thermocycler reaches the 15° hold, advance the thermocycler to the next step (94° for 2 min) and prepare the exonuclease mix:
  • thermocycler When the thermocycler reaches the 37° hold, add 1 ⁇ _ of exonuclease mix to each sample and then advance the thermocycler to the next step (37° for 30 min).
  • HCV genotype was confirmed by Versant HCV Genotyping Assay 2.0.
  • the 436 probes from tables 1-4 were used with Protocol 1 to target desired gene regions. Captured gene regions were sequenced using an Ion Torrent PGM and compared to sequences determined by Sanger
  • HCV probes from this invention correctly identified HCV-1 a and HCV-1 b viral variants compared to the Versant HCV genotyping assay. Resistance locus capture size averaged 200 bases, and read depth ranged between 50 to >50,000 fold. The probes detected mutations generating both nucleotide and amino acid polymorphisms. Figure 8 illustrates that among detected amino acid polymorphisms in our DAA-na ' ive clinical samples, we detected mutations reported to confer retroviral drug resistance in NS3, NS5a and NS5b proteins.
  • Selected observed mutations include: in NS3 - Q80L/K/R, D168G/E, I170T/V, 175L and E176G; in NS5a - M28T, Q30R, L31 M, P58S, Y58S and Y93H/N; and in NS5b - 71V, 1831, M414L/V, L419S, Y452H, V494A and V499A.
  • the probes agreed in 28/28 samples with Versant HCV genotyping assay in 1a/1 b clinical samples, see figure 9.
  • DxSeq detected mutations associated with resistance to antiviral drugs, such as TMC435, boceprevir, danoprevir, BI-201335, BMS-790052, GS-9190, BMS-650032, MK-3281 , VCH-916, and JTK-109.
  • Figures 11-13 describe the fraction of viral quasispecies represented by specific nucleic acid variants sequenced from selected samples, and illustrates detection of viral variants at 2% of the total viral nucleic acid present.
  • Sequencing reads from the 28 clinical samples in example 1 were analyzed to determine whether probe arms could be identified in the sequencing read. There were 13,583,863 reads for which at least the first probe arm could be identified uniquely (reads for which no probe arm can be identified are generally of poor quality and thus rejected). The probe arms were trimmed from these sequencing reads to yield only the capture regions. As the input files were FASTQ files containing both base calls and quality scores, both the nucleotides and quality scores were trimmed to produce a new FASTQ file. The resulting sequences were aligned against the human reference genome (hg19 from //genome. ucsc.edu) using the Bowtie2 alignment software version 2.0.0b6 and the following command line parameters: -q --sensitive --end-to- end -M 1 --no-unal --threads 8.
  • the resulting output will contain only sequences for which the probe capture can be mapped to one or more locations in the human genome using Bowtie2's sensitive alignment option. Only 71951 reads were mapped. Thus at most 0.53% of the sequencing reads that could be assigned to a probe contained a sequence of plausibly human origin.

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Abstract

L'invention concerne, entre autres, des sondes d'acides nucléiques, des mélanges de sondes, et des compositions contenant ces sondes, qui sont utiles dans la détection et la caractérisation du virus de l'hépatite, notamment des séquences d'acides nucléiques de l'hépatite C (VHC) présentes dans un échantillon pour essai, tel qu'un échantillon biologique provenant d'un patient. L'invention concerne également des méthodes de diagnostic et de traitement du VHC utilisant ces sondes, mélanges et compositions; ainsi que des systèmes pour mettre en oeuvre ces méthodes.
PCT/US2012/054901 2011-09-12 2012-09-12 Acides nucléiques pour détection multiplex du virus de l'hépatite c WO2013040060A2 (fr)

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WO2017021752A1 (fr) * 2015-08-03 2017-02-09 Universite Joseph Fourier Procédés d'amplification et de séquençage du génome d'un virus de l'hépatite c
WO2018108328A1 (fr) * 2016-12-16 2018-06-21 F. Hoffmann-La Roche Ag Procédé pour augmenter le débit d'un séquençage de molécule unique par concaténation de fragments d'adn court
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US20210292352A1 (en) * 2014-08-04 2021-09-23 The Trustees Of The University Of Pennsylvania Transcriptome In Vivo Analysis (TIVA) and Transcriptome In Situ Analysis (TISA)
US11873312B2 (en) * 2014-08-04 2024-01-16 The Trustees Of The University Of Pennsylvania Transcriptome in vivo analysis (TIVA) and transcriptome in situ analysis (TISA)
WO2017021752A1 (fr) * 2015-08-03 2017-02-09 Universite Joseph Fourier Procédés d'amplification et de séquençage du génome d'un virus de l'hépatite c
WO2017021471A1 (fr) * 2015-08-03 2017-02-09 Universite Grenoble Alpes Procédés d'amplification et de séquençage du génome d'un virus de l'hépatite c
WO2018108328A1 (fr) * 2016-12-16 2018-06-21 F. Hoffmann-La Roche Ag Procédé pour augmenter le débit d'un séquençage de molécule unique par concaténation de fragments d'adn court
CN110036117A (zh) * 2016-12-16 2019-07-19 豪夫迈·罗氏有限公司 通过多联短dna片段增加单分子测序的处理量的方法
JP2020501554A (ja) * 2016-12-16 2020-01-23 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 短いｄｎａ断片を連結することによる一分子シーケンスのスループットを増加する方法

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