WO2015187953A2

WO2015187953A2 - Method for direct microbial identification

Info

Publication number: WO2015187953A2
Application number: PCT/US2015/034202
Authority: WO
Inventors: Erik P. Johnson; Nik ISELY; Jamie L. Platt; Martin SIAW; Ron M. Kagan; Dale A. Schwab
Original assignee: Quest Diagnostics Investments LLC
Current assignee: Quest Diagnostics Investments LLC
Priority date: 2014-06-04
Filing date: 2015-06-04
Publication date: 2015-12-10
Anticipated expiration: 2016-12-04
Also published as: WO2015187953A8; US11421285B2; US20170191116A1; WO2015187953A3; US20230143248A1

Abstract

Described herein are methods for direct detection of microbial agent(s) in a polymicrobial sample, such as a biological sample from a human, without culturing the microbial agent(s). The direct detection can identify mixtures of bacteria and/or fungi in the sample. Also described are primer sequences and amplification techniques for performing the direct detection methods.

Description

METHOD FOR DIRECT MICROBIAL IDENTIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US provisional application no. 62/007,663, filed June 4, 2014, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] Methods for direct detection of microbial agent(s) in a sample, including a mixture of bacterial and fungal microbial agents, are disclosed. Nucleotide sequences and amplification techniques to identify microbial agent(s) in a sample also are described.

BACKGROUND OF THE INVENTION

[0003] The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

[0004] Microbial agents are currently identified by first culturing the agents using media and growth conditions, and then analyzing morphological/biochemical characteristics or DNA sequencing to determine their identity. Culturing isolates the microbial agents so they can be characterized by phenotypic or genotypic methods, and also provides conditions favorable to grow the microbial agents to produce enough material for analysis.

[0005] However, culturing has drawbacks. For example, culturing microbial agents is time consuming and not practical in situations where many different agents are presented in a sample. Moreover, recovering microbial agents from culture can be difficult if the culture conditions are not optimized, proper growth conditions are unknown, or certain agents are overgrown and mask the presence of slow-growing agents. The masking of some microbial agents can prevent correctly identifying all microbial agents in a sample.

[0006] The masking of some microbial agents is especially problematic with a biofilm sample (e.g., from a chronic wound, a catheter site infection, or due to periodontal disease) because multiple microbial agents can comprise the biofilm, but the most pathogenic specie(s) may be present in the lowest abundance. As a result, a patient's microbial infections are often treated with antibiotics that are not effective in treating their particular infection because the particular pathogenic species is unknown.

[0007] A technique to quickly identify all microbial agents in a sample would allow for quicker and more accurate identification of the source(s) of a microbial infection.

SUMMARY OF THE INVENTION

[0008] Provided herein are methods for determining the presence or absence of a microbial agent in a sample, comprising (a) contacting a sample containing sample nucleic acids with an amplification reaction mixture, wherein the amplification reaction mixture primers that specifically amplify at least one target sequence of bacterial 16S rDNA, at least one target sequence of fungal ITS rDNA, and at least one target sequence selected each of Mycobacterium rpoB, Staphylococcus rpoB, Streptococcus rpoB, Burkholderia recA, Enterococcus tuf, and Pseudomonas gvrB, the generate amplification reaction mixture containing the sample nucleic acids; (b) subjecting the amplification reaction mixture containing the sample nucleic acids to polymerase chain reaction (PCR) conditions to generate microbial amplicons; (c) producing adapter-tagged amplicons by attaching the microbial amplicons of step (b), if present, to nucleic acid adapters; (d) amplifying the adapter-tagged amplicons, if present, from step (c) to generate adapter-tagged amplicons; and (e) sequencing the adapter-tagged amplicons, if present, from step (c), wherein a microbial agent is determined to be present in the sample if a microbial amplicon is present and the sequence of the non-adapter portion of an adapter tagged microbial amplicon is at least 90% identical to a nucleotide fragment of bacterial 16S rDNA or fungal ITS rDNA. In some embodiments, the method further comprises identifying the species of bacteria and/or fungus in the sample as Mycobacterium, Staphylococcus, Streptococcus, Burkholderia, Enterococcus and/or Pseudomonas gvrB. In some embodiments, the reagent mixture further comprises a DNA polymerase and a plurality of free nucleotides comprising adenine, thymine, cytosine and guanine. In some embodiments, the PCR involves (i) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate the strands of the double stranded DNA from each other, (ii) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions to allow the first and second primers to hybridize with their complementary sequences on the first and second strands of the target DNA, and to allow Taq polymerase to extend the primers, and (iii) repeating steps (i) and (ii) at least 12 times to amplify microbial nucleic acids, if present, in the sample to produce microbial amplicons.

[0009] In some embodiments, BLAST (Basic Local Alignment Search Tool) is performed to make a broad identification based on the universal rDNA sequence followed by a BLAST of the taxon specific genes to provide resolution to species level.

[0010] In some embodiments, a post-extraction step is performed on the sample nucleic acids to remove human DNA prior to combining with the amplification reaction mixture.

[0011] In some embodiments, the amplification reaction mixture comprises primers comprising any of SEQ ID NOs 1-335. In some embodiments, multiple different target regions are amplified in a multiplexed reaction. In some embodiments, each target sequence amplification is performed in a separate, individual PCR reaction.

[0012] In some embodiments, primers that specifically amplify at least one target sequence of bacterial 16S rDNA comprise a sequence selected from among SEQ ID NOs 89- 103.

[0013] In some embodiments, primers that specifically amplify at least one target sequence of fungal ITS rDNA comprise a sequence selected from among SEQ ID NOs 119- 128.

[0014] In some embodiments, primers that specifically amplify at least one target sequence of Mycobacterium rpoB comprise a sequence selected from among SEQ ID NOs 139-152.

[0015] In some embodiments, primers that specifically amplify at least one target sequence of Streptococcus rpoB comprise a sequence selected from among SEQ ID NOs 181-233.

[0016] In some embodiments, primers that specifically amplify at least one target sequence of Staphylococcus rpoB comprise a sequence selected from among SEQ ID NOs 273-298.

[0017] In some embodiments, primers that specifically amplify at least one target sequence of Burkholderia recA comprise a sequence selected from among SEQ ID NOs 299- 306. [0018] In some embodiments, primers that specifically amplify at least one target sequence of Enterococcus tuf comprise a sequence selected from among SEQ ID NOs 307- 312.

[0019] In some embodiments, primers that specifically amplify at least one target sequence of Pseudomonas gvrB comprise a sequence selected from among SEQ ID NOs 313- 320.

[0020] In some embodiments, the primers further comprise a tag sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.

[0021] In some embodiments, the adapter sequence is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2.

[0022] In some embodiments, the adapters are attached via a primer comprising the adaptor sequence. In some embodiments, the primer comprising the adaptor sequence further comprises a multiplex identifier sequence. In some embodiments, the primer comprising the adaptor sequence further comprises a tag sequence specific for the microbial amplicon. In some embodiments, the tag sequence is selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.

[0023] In some embodiments, the adapters are attached via enzyme ligation.

[0024] In some embodiments, the sample nucleic acids are nucleic acids from a human biological sample. In some embodiments, the biological sample is a urine, sputum, vaginal fluid, sperm, blood or synovial fluid sample

[0025] Kits are also provided that comprise at least one of the oligonucleotide primers selected from the group consisting of SEQ ID NOs 1-335.

[0026] In some embodiments, the primers in a kit as disclosed herein further comprise a multiplex identifier sequence, a tag sequence and/or an adapter sequence. In some embodiments, one primer of a primer pair comprises an MID and both primers in a primer pair comprise adapter sequences. A forward primer and a reverse primer may comprise different adapter sequences. In some embodiments, the adapter sequence is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2. In some embodiments, the primers further comprise a multiplex identifier sequence. In some embodiments, the primers comprises a tag sequence specific for the microbial amplicon. In some embodiments, the tag sequence is selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4. BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 depicts direct detection of a microbial agent using primers comprising an adapter sequence (Adapter A or Adapter P) and a target specific sequence (SS), with or without a multiplex identifier (MID). Microbial amplicons are generated in a first amplification reaction (PCRl) using primers comprising a target specific sequence (SS). Adapters are attached in a second amplification reaction (PCR2) using the primers comprising an adapter sequence (Adapter A or Adapter P) and a target specific sequence (SS), with or without a multiplex identifier (MID).

[0028] Figure 2 depicts direct detection of a microbial agent using (i) primers comprising a target specific sequence (SS) and a tag (Tag) and (ii) primers comprising an adapter sequence (Adapter A or Adapter P) and the Tag sequence, with or without a multiplex identifier (MID). Microbial amplicons are generated in a first amplification reaction (PCRl) using primers comprising a target specific sequence (SS) and a tag (Tag). Adapters are attached in a second amplification reaction (PCR2) using the primers comprising an adapter sequence (Adapter A or Adapter P) and the Tag sequence, with or without a multiplex identifier (MID).

[0029] Figure 3 depicts direct detection of a microbial agent with bi-directional sequencing using (i) primers comprising a target specific sequence (SS) and a tag (Tag) and (ii) primers comprising an adapter sequence (Adapter A or Adapter P) and the Tag sequence, with or without a multiplex identifier (MID). Figure 3 differs from Figure 2 in that the adapters are attached in the opposite orientation.

[0030] Figure 4 depicts direct detection of a microbial agent using primers comprising a target specific sequence (SS) in a first amplification reaction (PCRl) and attaching a double stranded adapter sequence (Adapter A or Adapter P), with or without a multiplex identifier (MID), to the microbial amplicon using enzyme ligation (Apollo 324 Adapter Attachment). A second amplification reaction can be performed (PCRl) to further amplify the adapter-tagged amplicon.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS [0031] The term "amplify" as used herein with respect to nucleic acid sequences, refers to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods, such as PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al, Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam et al, Nucleic Acids Res. 2001 Jun 1;29(1 1):E54-E54; Hafner et al, Biotechniques 2001 Apr;30(4):852-6, 858, 860 passim; Zhong et al, Biotechniques 2001 Apr;30(4):852-6, 858, 860.

[0032] A "nucleic acid" as used herein refers to a nucleic acid that contains a sequence of a microbial gene, mRNA, cDNA or a portion of such a sequence. A nucleic acid may contain the coding region. A nucleic acid may be genomic DNA, cDNA, single stranded DNA or mRNA. In some embodiments, only a single strand of a sample nucleic acid is amplified and/or sequenced. In some embodiments both strands of double stranded DNA are amplified and sequenced. A nucleic acid may be present in a sample, such as a biological sample, or it may be isolated from the sample.

[0033] The term "sense strand" as used herein means the strand of double-stranded DNA (dsDNA) that includes at least a portion of a coding sequence of a functional protein. "Anti- sense strand" means the strand of dsDNA that is the reverse complement of the sense strand.

[0034] The terms "complementary" or "complementarity" as used herein with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refers to the base-pairing rules. The complement of a nucleic acid sequence as used herein refers to nucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel

association." For example, the sequence "5'-A-G-T-3"' is complementary to the sequence "3'-T-C-A-5." Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids described herein; these include, for example, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

[0035] The term "substantially complementary" as used herein means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences may comprise a contiguous sequence of bases that do not hybridize to a target sequence, positioned 3 Or 5' to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target sequence.

[0036] The term "hybridize" as used herein refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions.

Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length, more preferably 18-50 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions.

[0037] The term "stringent hybridization conditions" as used herein refers to

hybridization conditions at least as stringent as the following: hybridization in 50%

formamide, 5xSSC, 50 mM NaH₂P0₄, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42° C. overnight; washing with 2x SSC, 0.1 %> SDS at 45° C; and washing with 0.2x SSC, 0.1% SDS at 45° C. In another example, stringent hybridization conditions should not allow for hybridization of two nucleic acids which differ over a stretch of 20 contiguous nucleotides by more than two bases.

[0038] The term "dosage" or "gene dosage" refers to the number of copies of a gene, or portions of a gene, present in a sample. [0039] The term "primer" as used herein means a sequence of nucleic acid, including DNA, which hybridizes to a substantially complementary target sequence and is recognized by DNA polymerase to begin DNA replication. The term primer as used herein includes all forms of primers that may be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like.

[0040] The term "forward primer" as used herein means a primer that anneals to the anti- sense strand of dsDNA. A "reverse primer" anneals to the sense-strand of dsDNA.

[0041] The term "specific" as used herein in reference to an oligonucleotide primer means that the primer hybridization sequence of the primer has at least 12 bases of sequence identity with a portion of the nucleic acid to be amplified when the oligonucleotide and the nucleic acid are aligned. A primer that is specific for a nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and more preferably at least 98% sequence identity.

[0042] The term "flanking" as used herein with regard to primers means that a primer hybridizes to a target nucleic acid adjoining a region of interest sought to be amplified on the target. The skilled artisan will understand that preferred primers are pairs of primers that hybridize 5' from a region of interest, one on each strand of a target double stranded DNA molecule, such that nucleotides may be added to the 3' end of the primer by a suitable DNA polymerase. Primers that flank an exon are generally designed not to anneal to the exon sequence but rather to anneal to sequence that adjoins the exon (e.g., intron sequence).

However, in some cases, an amplification primer may be designed to anneal to the exon sequence.

[0043] "Sequencing depth" or "read depth" as used herein refers to the number of times a sequence has been sequenced (i.e., the depth of sequencing). As an example, read depth can be determined by aligning multiple sequencing run results and counting the start position of reads in nonoverlapping windows of a certain size (e.g., 100 bp). Copy number variation can be determined based on read depth using methods known in the art. For example, using a method described in Yoon et al, Genome Research 2009 September; 19(9): 1586-1592; Xie et al, BMC Bioinformatics 2009 Mar 6;10:80; or Medvedev et al, Nature Methods 2009 Nov;6(l 1 Suppl):S 13-20. Use of this type of method and analysis is referred to as a "read depth approach."

[0044] "Coverage depth" refers to the number of nucleotides from sequencing reads that are mapped to a given position.

[0045] The term "isolated" as used herein with respect to a nucleic acid (e.g., RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany such nucleic acid. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, oligonucleotides, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0046] The term "substantially pure" as used herein means a nucleic acid, represents more than 50% of the nucleic acid in a sample. The nucleic acid sample may exist in solution or as a dry preparation.

[0047] The term "coding sequence" as used herein means a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced there from.

[0048] The term "non-coding sequence" as used herein means a sequence of a nucleic acid or its complement, or a part thereof, which is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.

[0049] The term "about" as used herein means in quantitative terms plus or minus 10%.

METHODS

[0050] Described herein are methods for direct detection of one or more microbial agents (i.e., microbial agent(s)) in a sample. Direct detection refers to identifying microbial agent(s) in a sample without culturing the sample. Culturing as used herein refers to any technique in which microbial agents in a sample are sustained and/or expanded in vitro, for example, using media and/or growth conditions. In some embodiments, direct detection refers to identifying a mixture of different microbial agents in a sample, such as a mixture of different bacteria, a mixture of different fungi, and a mixture of bacterium/bacteria and fungus/fungi.

[0051] In some embodiments, methods for direct detection include extracting nucleic acid from a sample without separating different types of nucleic acid, such as nucleic acid from different types of microbial agents. In some embodiments, methods for direct detection include identifying microbial agent(s) in a sample after extracting nucleic acid from the sample. In specific embodiments, direct detection includes identifying microbial agent(s) in a mammalian biological sample, such as a human biological sample, after extracting nucleic acid from the sample. In other embodiments, direct detection includes identifying microbial agent(s) in a human biological sample after human nucleic acid has been separated and removed from extracted nucleic acid.

MICROBIAL AGENT

[0052] A microbial agent as used herein is any microorganism. In some embodiments, the microbial agent is a bacterium. In other embodiments, the microbial agent is a fungus. In some embodiments, the microbial agent is a species selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Burkholderia, Enterococcus, and

Pseufomonas.

[0053] A target sequence as described herein may represent one or more individual exon(s) or portion(s) of exon(s) of a microbial gene or one or more portions of a microbial mRNA. A target sequence also may include the promoter region and/or one or more introns of a microbial agent gene.

[0054] In some embodiments the target sequence represents the entire gene or the entire coding region. In some embodiments, the target sequence represents the entire coding region and at least one intron or a portion thereof and an adjacent region located immediately upstream (in the 5' direction) of the coding sequence. The adjacent, upstream region may consist of from about 100 nucleotides up to about 500, 750, 1000, 1100, or 1200 nucleotides of the sequence located immediately upstream of the coding sequence. In some

embodiments, the adjacent, upstream region comprises all or a portion of the promoter sequence.

SAMPLE [0055] A sample as used herein contains nucleic acid of microbial agent(s) in, or isolated from, any source. In some embodiments, the sample is a biological sample from a mammal. In specific embodiments, the mammal is a human.

[0056] In some embodiments, the biological sample is a body fluid or a tissue sample. In some embodiments the biological sample consists or comprises blood, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi, cultured cells, and combinations thereof.

[0057] In some embodiments, the biological sample is a fixed or frozen tissue. In some embodiments, the biological sample is whole blood of about 0.5 to 5 ml collected with EDTA, ACD or heparin as anti-coagulant. In some embodiments, the biological sample is amniotic fluid of 10-15 ml, cultured cells which are 80-100% confluent in two T-25 flasks, or 25 mg of chorionic villi.

[0058] In some embodiments, the sample contains one or more microbial agents. In some embodiments, the sample contains multiple microbial agents. In some embodiments, the sample contains a mixture of bacteria. In other embodiments, the sample contains a mixture of fungi. In other embodiments, the sample contains a mixture of bacterium/bacteria and fungus/fungi.

[0059] Processing methods to release or otherwise make available a nucleic acid for detection are well known in the art and may include steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of R A from a biological sample. In some embodiments, ) a sample taken from a patient is extracted using the MagNA Pure LC instrument or an equivalent tabletop instrument that performs rapid, cross-contamination- free preparation of nucleic acids and PCR setup. The instrument may utilize magnetic-bead technology and may be equipped with a robotic system and automatically isolates any type of nucleic acid. It further may be capable of processing up to 32 different samples in one batch.. The enables consistent isolation of high-quality DNA or RNA.

ADAPTER SEQUENCE

[0060] An adapter sequence (also referred to as a sequencing adapter) is ligated to the 5' end of the target specific sequence portion of the primer. This sequencing adapter is a short oligonucleotide of known sequence that can provide a priming site for both amplification and sequencing of the adjoining, unknown nucleic acid. As such, adapters allow binding of a fragment to a flow cell for high throughput, massively parallel sequencing, as described herein. Any adapter sequence may be included in a primer used in the present invention.

[0061] In some embodiments, all forward amplicons (i.e., amplicons extended from forward primers that hybridized with antisense strands of a target segment) contain the same adapter sequence. In some embodiments when double stranded sequencing is performed, all forward amplicons contain the same adapter sequence and all reverse amplicons (i.e., amplicons extended from reverse primers that hybridized with sense strands of a target segment) contain an adapter sequence that is different from the adapter sequence of the forward amplicons.

[0062] In some embodiments, the "forward" adapter sequence consists of or comprises: CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO: l) or a sequence 90%, 95%, 97%), 98%o, or 99% identical to SEQ ID NO: l . and the reverse adapter sequence consists of or comprises CCTCTCTATGGGCAGTCGGTGAT (SEQ ID NO:2) or a sequence 90%, 95%, 97%), 98%o, or 99% identical to SEQ ID NO:2. These sequences are provided in Table 1.

[0063] Other adapter sequences are known in the art. Some manufacturers recommend specific adapter sequences for use with the particular sequencing technology and machinery that they offer.

[0064] In some embodiments, when adapter-ligated and/or indexed primers are employed to amplify a target segment, the adapter sequence and/or index sequence gets incorporated into the amplicon (along with the target-specific primer sequence) during amplification. Therefore, the resulting amplicons are sequencing-competent and do not require the traditional library preparation protocol. Moreover, the presence of the index tag permits the differentiation of sequences from multiple sample sources.

[0065] In some embodiments, sequencing templates (amplicons) are prepared by emulsion-based clonal amplification of target segments using specialized fusion primers (containing an adapter sequence) and capture beads. A single adapter-bound fragment is attached to the surface of a bead, and an oil emulsion containing necessary amplification reagents is formed around the bead/fragment component. Parallel amplification of millions of beads with millions of single strand fragments produces a sequencer-ready library.

[0066] In some embodiments, the amplicons constituting the adapter-tagged (and, optionally, indexed) amplicon library are produced by polymerase chain reaction (PCR). In some embodiments, the amplicon library is generated using a multiplexed PCR approach, such as that disclosed in U.S. patent number 8,092,996, incorporated by reference herein in its entirety.

[0067] In other embodiments, each nucleic acid target segment may be amplified with non-adapter-ligated and/or non-indexed primers and a sequencing adapter and/or an index sequence may be subsequently ligated to each of the resulting amplicons.

[0068] In some embodiments, sequencing by ligation method using a DNA ligase is applied to determine the target sequence. This sequencing method relies on enzymatic ligation of oligonucleotides that are adjacent through local complementarity on a template DNA strand. This technology employs a partition of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated and the preferential ligation by DNA ligase for matching sequences results in a dinucleotide encoded color space signal at that position (through the release of a fluorescently labeled probe that corresponds to a known nucleotide at a known position along the oligo). This method can utilize Life Technologies' SOLiD™ sequencers.

MULTIPLEX IDENTIFIER

[0069] In some cases, amplicons from a single sample source further comprise an identical index sequence (also referred to as an index tag, a "barcode" or a multiplex identifier (MID)). In some cases, indexed amplicons are generated using primers (for example, forward primers and/or reverse primers) containing the index sequence. Such indexed primers may be included during library preparation as a "barcoding" tool to identify specific amplicons as originating from a particular sample source. Indexed amplicons from more than one sample source are quantified individually and then pooled prior to sequencing. As such, the use of index sequences permits multiple samples (i.e., samples from more than one sample source) to be pooled per sequencing run and the sample source subsequently ascertained based on the index sequence. Table 1 provides examples of MID sequences used in the methods described herein.

[0070] In some embodiments, amplicons from more than one sample source are pooled prior to high throughput sequencing. "Multiplexing" is the pooling of multiple adapter- tagged and indexed libraries into a single sequencing run. When indexed primer sets are used, this capability can be exploited for comparative studies. In some embodiments, amplicon libraries from up to 48 separate sources are pooled prior to sequencing. HIGH THROUGHPUT, MASSIVELY PARALLEL SEQUENCING

[0071] High throughput, massively parallel sequencing refers to sequencing methods that can generate multiple sequencing reactions of clonally amplified molecules and of single nucleic acid molecules in parallel. This allows increased throughput and yield of data. These methods are also known in the art as next generation sequencing (NGS) methods. NGS methods include, for example, sequencing-by-synthesis using reversible dye terminators, and sequencing-by-ligation.

[0072] In some embodiments, high throughput, massively parallel sequencing employs sequencing-by-synthesis with reversible dye terminators. In other embodiments, sequencing is performed via sequencing-by-ligation. In yet other embodiments, sequencing is single molecule sequencing.

[0073] Non-limiting examples of commonly used NGS platforms include Apollo 324™ NGS Library Prep System (IntengenX, Pleasanton, United States), Ion Torrent™ (Life Technologies, Carlsbad, CA), miRNA BeadArray (Illumina, Inc.), Roche 454™ GS FLX™- Titanium (Roche Molecular Diagnostics, Germany), and ABI SOLiD™ System (Applied Biosystems, Foster City, CA). Following the production of an adapter tagged and, optionally indexed, amplicon library, the amplicons are sequenced using high throughput, massively parallel sequencing.

KIT AND PRIMER(S

[0074] The direct detection methods as described herein can be performed using a kit comprising any one or more of the following components: universal primer(s) (e.g., 16S rDNA and ITS rDNA); primer(s), including primer(s) comprising one or more of a target specific sequence, adapter sequence, MID, and tag; dNTP; and other components for amplifying nucleic acid, such as by PCR (including via high throughput, massively parallel sequencing). In some embodiments, the kit comprises components to extract human nucleic acid from a sample.

[0075] In some embodiments, the kit comprises any one or more of SEQ ID NOs: 1-335, as listed in Tables 1-10. The kit can include a primer or primer pair comprising any combination of the sequences listed in Tables 1-10, with or without additional nucleic acid(s). For example, SEQ ID NO:23 is a primer consisting of SEQ ID NO: 1 (Adapter A sequence) and SEQ ID NO: 5 (MIDI). However, a primer or primer pair as described herein can include SEQ ID NO: l and SEQ ID NO:5 with additional nucleic acid(s) between the two sequences or flanking one or both sequences. In some embodiments, a primer or primer pair as described herein comprises a spacer between two or more of SEQ ID NOs: 1-335. Spacers are known in the art.

Table 1 : Adapter, Tag, and Multiplex Identifier Sequences

SEO ID Name Seauence

NO:

2 PGMA MID4 Comp CTACAGTGCT CTGAGTCGGAGACACGCAGGGATGAGATGG3 PGMA MID5 Comp CGTGTCTGAT CTGAGTCGGAGACACGCAGGGATGAGATGG4 PGMA MID6 Comp CTCGCGATAT CTGAGTCGGAGACACGCAGGGATGAGATGG5 PGMA MID7 Comp TAGAGACACG CTGAGTCGGAGACACGCAGGGATGAGATGG6 PGMA MID8 Comp GACACGCGAG CTGAGTCGGAGACACGCAGGGATGAGATGG7 PGMA MID9 Comp GCTGATACTA CTGAGTCGGAGACACGCAGGGATGAGATGG 8 PGMA MID 10 Comp CGCATAGAGA CTGAGTCGGAGACACGCAGGGATGAGATGG9 PGMA MIDI 1 Comp AGACGTATCA CTGAGTCGGAGACACGCAGGGATGAGATGG0 PGMA MID 12 Comp TAGCTCAGTA CTGAGTCGGAGACACGCAGGGATGAGATGG 1 PGMA MID 13 Comp CACTACTATG CTGAGTCGGAGACACGCAGGGATGAGATGG2 PGMA MID 14 Comp GTATCTCTCG CTGAGTCGGAGACACGCAGGGATGAGATGG3 PGMA MID 15 Comp TACGTCGTAT CTGAGTCGGAGACACGCAGGGATGAGATGG4 PGMA MID 16 Comp TAGTACGTGA CTGAGTCGGAGACACGCAGGGATGAGATGG

PCR2 Forward Primers (Primers with Adapter A, MID, and Forward Tag)5 PGMA MIDI FT CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGAGTGCGT

ACACTGACGACATGGTTCTACA

6 PGMA MID2 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGCTCGACA

ACACTGACGACATGGTTCTACA

7 PGMA MID3 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG AGACGCACTC

ACACTGACGACATGGTTCTACA

8 PGMA MID4 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG AGCACTGTAG

ACACTGACGACATGGTTCTACA

9 PGMA MID5 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG ATCAGACACG

ACACTGACGACATGGTTCTACA

0 PGMA MID6 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG ATATCGCGAG

ACACTGACGACATGGTTCTACA

1 PGMA MID7 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG CGTGTCTCTA

ACACTGACGACATGGTTCTACA

2 PGMA MID8 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG CTCGCGTGTC

ACACTGACGACATGGTTCTACA

3 PGMA MID9 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG TAGTATCAGC

ACACTGACGACATGGTTCTACA

4 PGMA MID 10 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG TCTCTATGCG

ACACTGACGACATGGTTCTACA

5 PGMA MIDI 1 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG TGATACGTCT

ACACTGACGACATGGTTCTACA

6 PGMA MID 12 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG TACTGAGCTA

ACACTGACGACATGGTTCTACA

7 PGMA MID 13 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG CATAGTAGTG

ACACTGACGACATGGTTCTACA

8 PGMA MID 14 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG CGAGAGATAC

ACACTGACGACATGGTTCTACA

9 PGMA MID 15 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG ATACGACGTA

ACACTGACGACATGGTTCTACA

0 PGMA MID 16 FT CCATCTCATCCCTGCGTGTCTCCGACTCAG TCACGTACTA

ACACTGACGACATGGTTCTACA

Primer with Adapter P and Reverse Tag

1 Primer P RT CCTCTCTATGGGCAGTCGGTGAT

TACGGTAGCAGAGACTTGGTCT

PCR2 Reverse Primers (Primers with Adapter A, MID, and Reverse Tag)2 CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGAGTGCGT

PGMA MIDI RT TACGGTAGCAGAGACTTGGTCT

3 CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGCTCGACA

PGMA MID2 RT TACGGTAGCAGAGACTTGGTCT

4 CCATCTCATCCCTGCGTGTCTCCGACTCAG AGACGCACTC

PGMA MID3 RT TACGGTAGCAGAGACTTGGTCT SEO ID Name Seauence

NO:

75 CCATCTCATCCCTGCGTGTCTCCGACTCAG AGCACTGTAG

PGMA MID4 RT TACGGTAGCAGAGACTTGGTCT

76 CCATCTCATCCCTGCGTGTCTCCGACTCAG ATCAGACACG

PGMA MID5 RT TACGGTAGCAGAGACTTGGTCT

77 CCATCTCATCCCTGCGTGTCTCCGACTCAG ATATCGCGAG

PGMA MID6 RT TACGGTAGCAGAGACTTGGTCT

78 CCATCTCATCCCTGCGTGTCTCCGACTCAG CGTGTCTCTA

PGMA MID7 RT TACGGTAGCAGAGACTTGGTCT

79 CCATCTCATCCCTGCGTGTCTCCGACTCAG CTCGCGTGTC

PGMA MID8 RT TACGGTAGCAGAGACTTGGTCT

80 CCATCTCATCCCTGCGTGTCTCCGACTCAG TAGTATCAGC

PGMA MID9 RT TACGGTAGCAGAGACTTGGTCT

81 CCATCTCATCCCTGCGTGTCTCCGACTCAG TCTCTATGCG

PGMA MID 10 RT TACGGTAGCAGAGACTTGGTCT

82 CCATCTCATCCCTGCGTGTCTCCGACTCAG TGATACGTCT

PGMA MID 11 RT TACGGTAGCAGAGACTTGGTCT

83 CCATCTCATCCCTGCGTGTCTCCGACTCAG TACTGAGCTA

PGMA MID 12 RT TACGGTAGCAGAGACTTGGTCT

84 CCATCTCATCCCTGCGTGTCTCCGACTCAG CATAGTAGTG

PGMA MID 13 RT TACGGTAGCAGAGACTTGGTCT

85 CCATCTCATCCCTGCGTGTCTCCGACTCAG CGAGAGATAC

PGMA MID 14 RT TACGGTAGCAGAGACTTGGTCT

86 CCATCTCATCCCTGCGTGTCTCCGACTCAG ATACGACGTA

PGMA MID 15 RT TACGGTAGCAGAGACTTGGTCT

87 CCATCTCATCCCTGCGTGTCTCCGACTCAG TCACGTACTA

PGMA MID 16 RT TACGGTAGCAGAGACTTGGTCT

Primer with Adapter P and Forward Tag

CCTCTCTATGGGCAGTCGGTGAT

88

Primer P FT ACACTGACGACATGGTTCTACA

Table 2: 16S Sequences

SEP Name Seauence

ID

NO:

105 ACACTGACGACATGGTTCTACA

Tag V2 F101b

GGCGAACGGGTGAGTAA

106 ACACTGACGACATGGTTCTACA

Tag V2 F101c

GGCGCACGGGTGAGTAA

107 ACACTGACGACATGGTTCTACA

Tag V2 F101d

GGCGGATGGGTGAGTAA

108 ACACTGACGACATGGTTCTACA

Tag V2 F101e

GGCAAACGGGTGAGTAA

109 ACACTGACGACATGGTTCTACA

Tag V2 F101f

GGCGAACGGGCGAGTAA

110 ACACTGACGACATGGTTCTACA

Tag V2 F101g

GGCGAACGGCTGAGTAA

111 TACGGTAGCAGAGACTTGGTCT

Tag V2 R356a CACTGCTGCCTCCCGTAG

112 TACGGTAGCAGAGACTTGGTCT

Tag V2 R356b TACTGCTGCCTCCCGTAG

PCR1 V3 (Tag, Sequence Specific)

113 ACACTGACGACATGGTTCTACA

Tag V3 F323a GACACGGTCCAGACTCCTAC

114 ACACTGACGACATGGTTCTACA

Tag V3 F323b GACACGGCCCAGACTCCTAC

115 ACACTGACGACATGGTTCTACA

Tag V3 F323c GACACGGTCCAAACTCCTAC

116 ACACTGACGACATGGTTCTACA

Tag V3 F323d GACACGGCCCAAACTCCTAC

117 ACACTGACGACATGGTTCTACA

Tag V3 F323e GATACGGCCCAGACTCCTAC

118 TACGGTAGCAGAGACTTGGTCT

Tag V3 R531a ATTACCGCGGCTGCTG

Table 3 : ITS Sequences

SEP Name Seauence

ID

NO:

AAACTCGGTCATTTAGAGGAAGTAA

134 ITSPGM2bP CCTCTCTATGGGCAGTCGGTGAT GCTGCGTTCTTCATCGATG

135 CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGAGTGCGT

ITSPGMF569A MIDI

ATCGAGTCTTTGAACGCACA

136 ITSPGMR820P CCTCTCTATGGGCAGTCGGTGAT CCTACCTGATCCGAGGTCAA

137 CCATCTCATCCCTGCGTGTCTCCGACTCAG ACGAGTGCGT

ITSPGMF570A MIDI

TCGAGTCTTTGAACGCACAT

138 ITSPGMR828P CCTCTCTATGGGCAGTCGGTGAT CGGGTATCCCTACCTGATCC

PCR1 (Tag, Sequence Specific)

335 ACACTGACGACATGGTTCTACA

Tag lTSlFb

AAACTCGGTCATTTAGAGGAAGTAA

336 Tag ITS2b TACGGTAGCAGAGACTTGGTCT GCTGCGTTCTTCATCGATG

337 Tag ITSF569 ACACTGACGACATGGTTCTACA ATCGAGTCTTTGAACGCACA

338 Tag ITSR820 TACGGTAGCAGAGACTTGGTCT CCTACCTGATCCGAGGTCAA able 4: Mycobacterium Sequences

SEP Name Seauence Description ID

NP:

Mycobacterium Fragment 1

139 MycoPGMF2649 GCAAGGTCACCCCGAAG

140 MycoPGMR2924 CGATGACGCCCTTGTTG

141 MycoPGMF2648 GGCAAGGTCACCCCGAAGG

142 MycoPGMR2934 AGGATCTTGCCGATGACG

Mycobacterium Fragment 2

143 MycoPGM2F2898 GACGCCACGGCAACAAG

144 MycoPGM2F2899 ACGCCACGGCAACAAG

145 MycoPGM2R3337 CAAGTGGTGCAGCTTCAGGATG Corynebacterium

146 MycoPGM2R3337d CARGTGGTGCAGCTTCAKGATG

147 MycoPGM2R3169 GGCGCCGTCGAACAC

148 MycoPGM2R3169d GGCRCCGTCGAACAC

149 MycoPGM2R3169a GGCACCGTCGAACAC

150 MycoPGM2R3169b GGCGCCGTCGAACAC

Mycobacterium Fragment 3

151 MycoPGM2F3148 CACCCCGGTGTTCGAC

152 MycoPGM2R3391 CTGGGTGATCATCGAGTACG

Fragment 1 Forward Reading Set (Adapter A or Adapter P)

153 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGMF2649A

GCAAGGTCACCCCGAAG

154 CCTCTCTATGGGCAGTCGGTGAT

MycoPGMR2924P

CGATGACGCCCTTGTTG

155 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGMF2648A

GGCAAGGTCACCCCGAAGG

156 CCTCTCTATGGGCAGTCGGTGAT

MycoPGMR2934P

AGGATCTTGCCGATGACG

157 MycoPGMF2649A CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT GCAAGGTCACCCCGAAG

Fragment 1 Reverse Reading Set (Adapter P or Adapter A)

158 CCTCTCTATGGGCAGTCGGTGAT

MycoPGMF2649P

GCAAGGTCACCCCGAAG

159 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGMR2924A

CGATGACGCCCTTGTTG SEP Name Seauence Description ID

NO:

160 CCTCTCTATGGGCAGTCGGTGAT

MycoPGMF2648P

GGCAAGGTCACCCCGAAGG

161 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGMR2934A

AGGATCTTGCCGATGACG

Fragment 2 Forward Reading Set (Adapter A or Adapter P)

162 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2F2898A

GACGCCACGGCAACAAG

163 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2F2899A

ACGCCACGGCAACAAG

164 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2R3337P

CAAGTGGTGCAGCTTCAGGATG

165 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2R3337dP

CARGTGGTGCAGCTTCAKGATG

166 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2R3169P

GGCGCCGTCGAACAC

167 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2R3169dP

GGCRCCGTCGAACAC

168 MycoPGM2F2898A CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT GACGCCACGGCAACAAG

169 MycoPGM2F2899A CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT ACGCCACGGCAACAAG

Fragment 2 Reverse Reading Set (Adapter P or Adapter A)

170 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2F2898P

GACGCCACGGCAACAAG

171 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2F2899P

ACGCCACGGCAACAAG

172 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2R3337A

CAAGTGGTGCAGCTTCAGGATG

173 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2R3337dA

CARGTGGTGCAGCTTCAKGATG

174 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2R3169A

GGCGCCGTCGAACAC

175 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2R3169dA

GGCRCCGTCGAACAC

Fragment 3 Forward Reading Set (Adapter A or Adapter P)

176 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2F3148A

CACCCCGGTGTTCGAC

177 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2R3391P

CTGGGTGATCATCGAGTACG

178 MycoPGM2F3148A CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT CACCCCGGTGTTCGAC

Fragment 3 Reverse Reading Set (Adapter P or Adapter A)

179 CCTCTCTATGGGCAGTCGGTGAT

MycoPGM2F3148P

CACCCCGGTGTTCGAC

180 CCATCTCATCCCTGCGTGTCTCCGACTCAG

MycoPGM2R3391A

CTGGGTGATCATCGAGTACG

Table 5 : Streptococcus Sequences

SEP Name Seauence Description ID

NO:

oralis suis anginosus

182 pyog dysgalac

StrepF 1475b CCTTAGGACCTGGTGGTT canis equi agalac pneumo sang

183 StrepF1475c GCTTTAGGTCCTGGTGGTT mutans

184 StrepF 1475d CCTTGGGGCCTGGTGGTT mitisB

185 StrepF 1475e CCTTAGGGCCTGGTGGTT Parasanguinis

186 saliv therm pyog

StrepR1720a CTTCTTCGTCGGCAGTCAAC

canis

187 pyog2 agalac

StrepR1720b CTTCTTCATCAGCAGTCAACC

dysgalac

188 StrepR1720c CTTCTTCATCAGCAGTTAGC equi

189 StrepR1720d CTTCTTCATCAGCAGTAAGC mutans

190 StrepR1720e CTTCTTCATCAGCTGTCAAC pneumo

191 mitis oralis paras

StrepR1720f CTTCTTCATCGGCTGTCAAC

suis

192 StrepR1720g CTTCCTCGTCAGCGGTCAAC sang

193 StrepR1720h CTTCTTCGTCCGCTGTCAGC anginosus

194 StrepR1720i CTTCTTCATCCGCTGTTAGC intermedius

Streptococcus Fragment 2

195 StrepF 1875a TGCGACAGCATGTATTCCTT

196 StrepF 1875b CGCAACAGCATGTATTCCTT agalac

197 pyogenes dysgalac

StrepF 1875c TGCAACGGCATGTATTCCTT

canis

198 StrepF 1875d GGCAACGGCATGTATTCCTT intermedius

199 saliv therm pyog

StrepR2148a TGAGTTTGAACGACGGAATTT dysgalac pneumo mitisB paras

200 StrepR2148b TGAGTTGGAGCGACGGAATTT canis

201 StrepR2148c AGAGTTTGAACGGCGGAATTT equi anginosus

202 StrepR2148d AGAGTTAGAACGACGGAATTT mutans

203 StrepR2148e TGAGTTTGAACGGCGGAATTT agalactie

204 StrepR2148f TGAGTTAGAACGACGGAATTT mitis oralis

205 StrepR2148g TGAGTTAGAACGGCGGAATTT sang intermedius

Streptococcus Fragment 3

206 StrepF2885a TGAACATCGGTCAGGTTATGG salivarus suis

207 thermo dysgalac

StrepF2885b TGAACATTGGTCAGGTTATGG

sanguin

208 StrepF2885c TGAATATTGGTCAGGTTATGG pyogenes

209 pneumo mitis

StrepF2885d TGAATATCGGTCAGGTTATGG

oralis paras

210 StrepF2885e TGAACATCGGACAAGTTATGG canis

211 StrepF2885f TGAACATTGGACAGGTTATGG equi

212 StrepF2885g TGAACATTGGGCAAGTTATGG mutans

213 StrepF2885h TGAATATCGGACAAGTTATGG agalac intermedius

214 StrepF2885i TGAATATTGGTCAAGTTATGG anginosus

215 salivarus thermo

StrepR3134a TGAAGTTTATCATCAACCATGTG pyog dysgal canis suis

216 StrepR3134b TGCAATTTATCATCAACCATGTG mutans mitis oralis

217 StrepR3134c TGCAACTTATCATCAACCATGTG agalac

218 StrepR3134d TGAAGCTTATCATCTACCATGTG intermedius

219 StrepR3134e TGGAGTTTATCATCTACCATGTG sang

220 StrepR3134f TGAAGCTTATCATCAACCATGTG equi

SEP Name Seauence Description ID

NO:

254 StrepPGMR3134a CCTCTCTATGGGCAGTCGGTGAT

255 StrepPGMR3134b CCTCTCTATGGGCAGTCGGTGAT

256 StrepPGMR3134c CCTCTCTATGGGCAGTCGGTGAT

257 StrepPGMR3134d CCTCTCTATGGGCAGTCGGTGAT

258 StrepPGMR3134e CCTCTCTATGGGCAGTCGGTGAT

259 StrepPGMR3134f CCTCTCTATGGGCAGTCGGTGAT

260 StrepPGMR3134g CCTCTCTATGGGCAGTCGGTGAT

261 StrepPGMR3134h CCTCTCTATGGGCAGTCGGTGAT

PGM Fragment 4 Set (Adapter A, MID or Adapter P)

262 StrepPGMF3106a CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT

263 StrepPGMF3106b CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT

264 StrepPGMF3106c CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT

265 StrepPGMF3106d CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT

266 StrepPGMF3106e CCATCTCATCCCTGCGTGTCTCCGACTCAG

MIDI ACGAGTGCGT

267 StrepPGMR3366a CCTCTCTATGGGCAGTCGGTGAT

268 StrepPGMR3366b CCTCTCTATGGGCAGTCGGTGAT

269 StrepPGMR3366c CCTCTCTATGGGCAGTCGGTGAT

270 StrepPGMR3366d CCTCTCTATGGGCAGTCGGTGAT

271 StrepPGMR3366e CCTCTCTATGGGCAGTCGGTGAT

272 StrepPGMR3366f CCTCTCTATGGGCAGTCGGTGAT

Table 6: Staphylococcus Sequences

SEP Name Seauence Description ID

NO:

291 StaphR1715c ACATAGCTATCCTCTTCATCAGC epidermidis

292 StaphR1715d ACATAGCTATCTTCTTCGTCAGC aureus

293 StaphR1715e ACATAACTGTCTTCTTCATCAGC lugdunensis

Staphylococcus Fragment 4

294 StaphF3224a TCGGTGAGATGGAGGTATGG

295 StaphF3224b TCGGTGAGATGGAAGTATGG lugdunensis

296 StaphF3224c TCGGTGAAATGGAAGTATGG saprophyticus

297 StaphR3388a CTCGGAATGATTCTGGAACAC

298 intermedius, capitis,

StaphR3388b CTCGGAATGATTCAGGAACAC

lugdunensis, saprophyticus

Table 7: Burkholderia Sequences

Table 8: Enterococcus Sequences

Table 9: Pseudomonas Sequences

SEP Name Seauence

ID

NO:

319 gyrbF2 GTGCTTTACCAACAACATCCCTCA

320 gyrbR2 TGTCTTTGGTCTGGGAGCTGAAC

Table 10: IDT Label Sequences

[0076] The following examples serve to illustrate the present invention. The examples are in no way intended to limit the scope of the invention.

EXAMPLE 1 : Direct Detection Using Primers With Adapter Sequence and Target Specific Sequence

[0077] A biological sample is obtained from a human individual and nucleic acid extracted using the MagNA Pure LC instrument (Roche Molecular Diagnostics, Germany). A post-extraction step is performed to remove human nucleic acid from the sample. [0078] The remaining nucleic acid from the sample is amplified using universal 16S rDNA and ITS rDNA primers. The amplification is performed using PCR. The resulting amplified nucleic acid is then amplified again using PCR with bacterial or fungal specific DNA oligonucleotide primer pairs.

[0079] Next, primer pairs comprising both a target specific sequence (e.g., specific for a particular sequence within a microbial gene) and an adapter sequence are used to perform a third amplification process. The forward and reverse primers in the primer pairs contain different adapter sequences. The primers can optionally include a MID. This process attaches the adapter sequences to the microbial nucleic acid.

[0080] The amplicons are then sequenced using a high throughput, massively parallel platform to identify the nucleic acid sequence of the microbial agent(s) in the sample. The sequences are compared against a BLAST of the rDNA targets to identify the specific microbial agent(s) present in the sample.

EXAMPLE 2: Direct Detection Using Primers With Adapter Sequence and Multiplex Identifier

[0081] A biological sample is obtained from a human individual and nucleic acid extracted using the MagNA Pure LC instrument (Roche Molecular Diagnostics, Germany). A post-extraction step is performed to remove human nucleic acid from the sample.

[0082] The remaining nucleic acid from the sample is amplified using universal 16S rDNA and ITS rDNA primers. The amplification is performed using PCR. The resulting amplified nucleic acid is then amplified again using PCR with bacterial or fungal specific DNA oligonucleotide primer pairs comprising a target specific sequence and a tag.

[0083] Next, primer pairs comprising an adapter sequence and a MID are used to perform a third amplification process, in which the MID hybridizes to the tag from the second amplification process. The forward and reverse primers in the primer pairs contain different adapter sequences. This process attaches the adapter sequence to the microbial nucleic acid.

[0084] The amplicons are then sequenced using a high throughput, massively parallel platform to identify the nucleic acid sequence of the microbial agent(s) in the sample. The sequences are compared against a BLAST of the rDNA targets to identify the specific microbial agent(s) present in the sample. [0085] Alternatively, bi-directional sequencing can be performed, in which the forward and reverse primers in each primer pair have the opposite adapter sequence attached thereto.

EXAMPLE 3: Direct Detection Using Enzyme Ligation To Attach Adapter

[0086] A biological sample is obtained from a human individual and nucleic acid extracted using the MagNA Pure LC instrument (Roche Molecular Diagnostics, Germany). A post-extraction step is performed to remove human nucleic acid from the sample.

[0087] The remaining nucleic acid from the sample is amplified using universal 16S rDNA and ITS rDNA primers. The amplification is performed using PCR. The resulting amplified nucleic acid is then amplified again using PCR with bacterial or fungal specific DNA oligonucleotide primer pairs.

[0088] Next, enzyme ligation is performed to attached a nucleotide comprising an adapter sequence and MID to the microbial nucleic acid. The resulting adapter-tagged microbial nucleic acid is then amplified using a primer pair to produce amplicons.

[0089] The amplicons are then sequenced using a high throughput, massively parallel platform to identify the nucleic acid sequence of the microbial agent(s) in the sample. The sequences are compared against a BLAST of the rDNA targets to identify the specific microbial agent(s) present in the sample.

EXAMPLE 4: Direct Identification of Different Microbial Species in Polymicrobial Samples

[0090] A -459 bp segment of the V3-V4 bacterial 16s rrna gene was amplified with target- specific PCR primers with 5' overhang adapters. The amplification mix contained the following ingredients in sufficient volume for a quarter plate and a half plate.

[0091] Index sequences and adapters were ligated to the 5' and 3' ends of the amplicons to allow for paired end sequencing. The library derived from 15 samples was normalized and pooled, and loaded onto a MiSeq® sequencer for clustering and paired-end sequencing with the 250 bp paired end sequencing chemistry and a nano-flow cell.

[0092] Paired-end reads were merged and quality-filtered. Sequences were dereplicated, singletons were discarded, and then sequences clustered into centroids with a radius of 2%. Operational taxonomic units (OTUs) constructed from the centroids for each sample were searched against the Living Tree Program database release 111, available at http://www.arb- silva.de/projects/living-tree/ and/or the NCBI 16S rrna sequence database. Species identifications and the relative abundance of each identified species in the samples tested were tabulated.

[0093] Results

[0094] Sequence metrics

[0095] 421,105 raw reads were obtained, 368,337 reads passed the quality filtering stage for a PF rate of 87.5%. 93% of reads had a median Q value >Q30. The read distribution was normally distributed between the 15 samples in the pooled library with 4.1% ± 1.9% (1 SD) reads per sample. The negative control did not have an appreciable number of detectable reads (Table 11).

[0096] The vast majority of merged paired end reads produced full length amplicon sequence of 465 bp, or 427 bp after the target-specific PCR primers were trimmed.

Table 11: Reads and Operational Taxonomies Units (OTUs) per Sample reads

% of PF OTUs

Sample Description clustered

reads (>0.5%)

in OTUs

Ml mixed organisms 4.37 13681 7

M2 mixed organisms 4.05 1 1764 14

M3 mixed organisms 6.71 19967 8

M4 mixed organisms 3.65 10087 1 1

M5 mixed organisms 7.77 19821 8

M6 mixed organisms 3.30 7232 6

SI pure sample 5.55 19673 1

S2 pure sample 2.51 8874 1 S3 pure sample 4.39 15577

S4 pure sample 2.27 8073

S5 pure sample 6.78 23824

S6 pure sample 3.05 10899

PI patient sample 2.78 9731

P2 patient sample 1.46 5147

P3 patient sample 2.21 7705

NEG Negative Ctrl 0.007 NA

Organism Identification is shown in Table 12 below:

Table 12: Identification of Pure (S1-S6) and Patient (P1-P3) Samp!

Align % of Reads

Sample Identification (bp) Match Reads (N) Experimental Input

P1 S13 Pseudomonas_aeruginosa 425 100% 100% 9731 Pseudomonas aeruginosa

P2_S14 Neisseria_sicca 425 99.5% 100% 5147 Neisseria sicca

P3_S15 Bacillus_cereus 425 100% 100% 7705 Bacillus cereus

S1_S7 Neisseria lactamica 425 100% 100% 19673 Neisseria lactamica

S2_S8 Acinetobacter baumannii 425 100% 100% 8874 Acinetobacter baumannii

S3 S9 Salmonella enterica 425 100% 69.0% 10753

Salmonella cholereasuis

S3 S9 Enterobacter cloacae 425 99.5% 31.0% 4824

S4_S10 E. fergusonii/E. coli/Shigella 425 100% 100% 8073 Shigella sonnei

S5_S11 Bordetella_parapertussis 425 100% 100% 23824 Bordetella parapertussis

S6_S12 E. fergusonii/E. coli/Shigella 425 100% 100% 10899 E. coli

[0098] The bioinformatic pipeline successfully identified the input species in the 6 pure samples and 3 patient samples (Table 12). Sample 3 appeared to contain a mixture of two species at a 2: 1 ratio. The origin of the second species (E. cloacae) is not known.

[0099] Table 13 below demonstrates successful recovery of most input species from the mixed samples. E. cloacae, used in samples Ml and M2, appeared as a contaminant in

samples M3 and M5. Table 13: Identification of Six Mixed Bacterial Samples

[00100] These results demonstrate that the methods of the present application, which generate high quality paired-end sequence reads for sequence fragments of short length (in this case a 427 bp was used), accurately identify bacterial species in polymicrobial samples through rDNA amplification and sequencing.

EXAMPLE 5: Direct Identification of Different Microbial Species in Polymicrobial Samples from Subjects Affected by Infections Difficult to Diagnose

[00101] Biological fluids, including urine, sputum, vaginal fluid, sperm, blood and synovial fluid are collected from subjects affected by infections that are difficult to diagnose. The subjects are affected by chronic wound infections, lung infections, urinary tract infections, vaginal infections or infections of otherwise sterile body sites or of prosthetic implants. The samples are directly analyzed for the presence of gram-positive and gram- negative bacterial species without the need for culturing the bacterial colonies.

[00102] Results

[00103] Organisms that constitute 10% or more of a mixed population of three or more bacterial species that are present in the fluid sample are detected by 16s rDNA as described in Example 4 above.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for determining the presence or absence of one or more microbial agents in a polymicrobial sample without culturing the microbial agents, comprising

(a) contacting a sample containing sample nucleic acids with an amplification reaction mixture, wherein the amplification reaction mixture primers that specifically amplify at least one target sequence of bacterial 16S rDNA, at least one target sequence of fungal ITS rDNA, and at least one target sequence selected each of Mycobacterium rpoB, Staphylococcus rpoB, Streptococcus rpoB, Burkholderia recA, Enterococcus tuf, and Pseudomonas gvrB, the generate an amplification reaction mixture containing the sample nucleic acids;

(b) subjecting the amplification reaction mixture containing the sample nucleic acids of step (a) to polymerase chain reaction (PCR) conditions to generate microbial amplicons;

(c) producing adapter-tagged amplicons by attaching each of the microbial amplicons of step (b), if present, to nucleic acid adapters,

(d) amplifying the adapter-tagged amplicons, if present, from step (c) to generate adapter-tagged amplicons; and

(e) sequencing the adapter-tagged amplicons, if present, from step (d) wherein a microbial agent is determined to be present in the sample if a microbial amplicon is present and the sequence of the non-adapter portion of an adapter tagged microbial amplicon is at least 90% identical to a nucleotide fragment of bacterial 16S rDNA or fungal ITS rDNA.

2. The method of claim 1, wherein the method further comprises identifying the species of bacteria and/or fungus in the sample as Mycobacterium, Staphylococcus,

Streptococcus, Burkholderia, Enterococcus and/or Pseudomonas gvrB.

3. The method of claim 1, wherein BLAST alignment analysis is performed to identify the species of bacteria and/or fungi present in the sample.

4. The method of claim 1, wherein a post-extraction step is performed on the sample nucleic acids to remove human DNA prior to combining with the amplification reaction mixture.

5. The method of claim 1, wherein the amplification reaction mixture of comprises

primers comprising any of SEQ ID NOs 1-335.

6. The method of claim 1 wherein each target region is amplified in a multiplexed

reaction.

7. The method of claim 1 wherein each target region is amplified in a separate, individual PCR reaction.

8. The method of claim 1, wherein the sample is a biological sample.

9. The method of claim 8, wherein the biological sample is a urine, sputum, vaginal fluid, sperm, blood or synovial fluid sample.

10. The method of claim 1, wherein

(a) the primers that specifically amplify at least one target sequence of bacterial 16S rDNA comprise a sequence selected from among SEQ ID NOs 89-103;

(b) the primers that specifically amplify at least one target sequence of fungal ITS rDNA comprise a sequence selected from among SEQ ID NOs 119-128;

(c) the primers that specifically amplify at least one target sequence of Mycobacterium rpoB comprise a sequence selected from among SEQ ID NOs 139-152;

(d) the primers that specifically amplify at least one target sequence of Streptococcus rpoB comprise a sequence selected from among SEQ ID NOs 181-233;

(e) the primers that specifically amplify at least one target sequence of Staphylococcus rpoB comprise a sequence selected from among SEQ ID NOs 273-298;

(f) the primers that specifically amplify at least one target sequence of Burkholderia recA comprise a sequence selected from among SEQ ID NOs 299-306; (g) the primers that specifically amplify at least one target sequence of Enterococcus tuf comprise a sequence selected from among SEQ ID NOs 307-312; and/or

(h) the primers that specifically amplify at least one target sequence of Pseudomonas gvrB comprise a sequence selected from among SEQ ID NOs 313-320.

11. The method of claim 1 , wherein the primers further comprise a tag sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:4.

12. The method of claim 1, wherein the adapter sequence is selected from the group

consisting of SEQ ID NO: l and SEQ ID NO:2.

13. The method of claim 1, wherein the adapters are attached via a primer comprising the adaptor sequence.

14. The method of claim 13, wherein the primer comprising the adaptor sequence further comprises a multiplex identifier sequence.

15. The method of claim 13, wherein the primer comprising the adaptor sequence further comprises a tag sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:4.

16. A kit comprising at least one oligonucleotide primer selected from the group consisting of SEQ ID NOs 1-335.

17. The kit of claim 16, wherein the kit wherein the primers further comprise an adapter sequence.

18. The kit of claim 17, wherein the adapter sequence is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2.

19. The kit of claim 15, wherein the primers further comprise a multiplex identifier

sequence.

20. The kit of claim 15, wherein the primers further comprise a tag sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.