Patent Publication Number: US-2023143248-A1

Title: Method for direct microbial identification

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. provisional application No. 62/007,663, filed Jun. 4, 2014, the contents of which are incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     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 
     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. 
     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. 
     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. 
     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&#39;s microbial infections are often treated with antibiotics that are not effective in treating their particular infection because the particular pathogenic species is unknown. 
     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 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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 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. 
     In some embodiments, the adapters are attached via enzyme ligation. 
     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 
     Kits are also provided that comprise at least one of the oligonucleotide primers selected from the group consisting of SEQ ID NOs 1-335. 
     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 
         FIG.  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 (PCR1) 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). 
         FIG.  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 (PCR1) 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). 
         FIG.  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).  FIG.  3    differs from  FIG.  2    in that the adapters are attached in the opposite orientation. 
         FIG.  4    depicts direct detection of a microbial agent using primers comprising a target specific sequence (SS) in a first amplification reaction (PCR1) 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 (PCR1) to further amplify the adapter-tagged amplicon. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     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 April; 30(4):852-6, 858, 860 passim; Zhong et al., Biotechniques 2001 April; 30(4):852-6, 858, 860. 
     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. 
     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. 
     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. 
     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. 
     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 &amp; Sons, Secaucus, N.J. In some embodiments, specific hybridization occurs under stringent hybridization conditions. 
     The term “stringent hybridization conditions” as used herein refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5×SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart&#39;s solution at 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with 0.2×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. 
     The term “dosage” or “gene dosage” refers to the number of copies of a gene, or portions of a gene, present in a sample. 
     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. 
     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. 
     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. 
     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. 
     “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 November; 6(11 Suppl):S13-20. Use of this type of method and analysis is referred to as a “read depth approach.” 
     “Coverage depth” refers to the number of nucleotides from sequencing reads that are mapped to a given position. 
     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. 
     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. 
     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&#39;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. 
     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. 
     The term “about” as used herein means in quantitative terms plus or minus 10%. 
     Methods 
     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. 
     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 
     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  Pseudomonas.    
     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. 
     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 
     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. 
     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. 
     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. 
     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. 
     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 RNA 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 
     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. 
     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. 
     In some embodiments, the “forward” adapter sequence consists of or comprises: CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO:1) or a sequence 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1. and the reverse adapter sequence consists of or comprises CCTCTCTATGGGCAGTCGGTGAT (SEQ ID NO:2) or a sequence 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:2. These sequences are provided in Table 1. 
     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. 
     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. 
     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. 
     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. Pat. No. 8,092,996, incorporated by reference herein in its entirety. 
     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. 
     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&#39; SOLiD™ sequencers. 
     Multiplex Identifier 
     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. 
     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 
     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. 
     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. 
     Non-limiting examples of commonly used NGS platforms include Apollo 324™ NGS Library Prep System (IntengenX, Pleasanton, United States), Ion Torrent™ (Life Technologies, Carlsbad, Calif.), miRNA BeadArray (Illumina, Inc.), Roche 454™ GS FLX™-Titanium (Roche Molecular Diagnostics, Germany), and ABI SOLiD™ System (Applied Biosystems, Foster City, Calif.). 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) 
     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. 
     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 (MID1). However, a primer or primer pair as described herein can include SEQ ID NO:1 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 
               
            
           
           
               
               
               
            
               
                 SEQ ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
            
           
           
               
            
               
                 Adapter Sequences 
               
            
           
           
               
               
               
            
               
                  1 
                 Adapter A 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
               
               
                  2 
                 Adapter P 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
               
            
           
           
               
            
               
                 Tag Sequences 
               
            
           
           
               
               
               
            
               
                  3 
                 Forward Tag 
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                  4 
                 Reverse Tag 
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
            
           
           
               
            
               
                 Multiplex Index Sequences 
               
            
           
           
               
               
               
            
               
                  5 
                 MID1 
                 ACGAGTGCGT 
               
               
                   
               
               
                  6 
                 MID2 
                 ACGCTCGACA 
               
               
                   
               
               
                  7 
                 MID3 
                 AGACGCACTC 
               
               
                   
               
               
                  8 
                 MID4 
                 AGCACTGTAG 
               
               
                   
               
               
                  9 
                 MID5 
                 ATCAGACACG 
               
               
                   
               
               
                 10 
                 MID6 
                 ATATCGCGAG 
               
               
                   
               
               
                 11 
                 MID7 
                 CGTGTCTCTA 
               
               
                   
               
               
                 12 
                 MID8 
                 CTCGCGTGTC 
               
               
                   
               
               
                 13 
                 MID9 
                 TAGTATCAGC 
               
               
                   
               
               
                 14 
                 MID10 
                 TCTCTATGCG 
               
               
                   
               
               
                 15 
                 MID11 
                 TGATACGTCT 
               
               
                   
               
               
                 16 
                 MID12 
                 TACTGAGCTA 
               
               
                   
               
               
                 17 
                 MID13 
                 CATAGTAGTG 
               
               
                   
               
               
                 18 
                 MID14 
                 CGAGAGATAC 
               
               
                   
               
               
                 19 
                 MID15 
                 ATACGACGTA 
               
               
                   
               
               
                 20 
                 MID16 
                 TCACGTACTA 
               
               
                   
               
               
                 21 
                 MID17 
                 CGTCTAGTAC 
               
               
                   
               
               
                 22 
                 MID18 
                 TCTACGTAGC 
               
               
                   
               
            
           
           
               
            
               
                 Primers with Adapter and Multiplex Index Sequences 
               
            
           
           
               
               
               
            
               
                 23 
                 PGMA MID1 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGAGTGCGT 
               
               
                   
               
               
                 24 
                 PGMA MID2 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGCTCGACA 
               
               
                   
               
               
                 25 
                 PGMA MID3 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGACGCACTC 
               
               
                   
               
               
                 26 
                 PGMA MID4 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGCACTGTAG 
               
               
                   
               
               
                 27 
                 PGMA MID5 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATCAGACACG 
               
               
                   
               
               
                 28 
                 PGMA MID6 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATATCGCGAG 
               
               
                   
               
               
                 29 
                 PGMA MID7 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGTGTCTCTA 
               
               
                   
               
               
                 30 
                 PGMA MID8 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CTCGCGTGTC 
               
               
                   
               
               
                 31 
                 PGMA MID9 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TAGTATCAGC 
               
               
                   
               
               
                 32 
                 PGMA MID10 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCTCTATGCG 
               
               
                   
               
               
                 33 
                 PGMA MID11 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TGATACGTCT 
               
               
                   
               
               
                 34 
                 PGMA MID12 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TACTGAGCTA 
               
               
                   
               
               
                 35 
                 PGMA MID13 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CATAGTAGTG 
               
               
                   
               
               
                 36 
                 PGMA MID14 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGAGAGATAC 
               
               
                   
               
               
                 37 
                 PGMA MID15 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATACGACGTA 
               
               
                   
               
               
                 38 
                 PGMA MID16 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCACGTACTA 
               
               
                   
               
               
                 39 
                 PGMA MID1 Comp 
                 ACGCACTCGT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 40 
                 PGMA MID2 Comp 
                 TGTCGAGCGT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 41 
                 PGMA MID3 Comp 
                 GAGTGCGTCT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 42 
                 PGMA MID4 Comp 
                 CTACAGTGCT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 43 
                 PGMA MID5 Comp 
                 CGTGTCTGAT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 44 
                 PGMA MID6 Comp 
                 CTCGCGATAT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 45 
                 PGMA MID7 Comp 
                 TAGAGACACG CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 46 
                 PGMA MID8 Comp 
                 GACACGCGAG CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 47 
                 PGMA MID9 Comp 
                 GCTGATACTA CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 48 
                 PGMA MID10 Comp 
                 CGCATAGAGA CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 49 
                 PGMA MID11 Comp 
                 AGACGTATCA CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 50 
                 PGMA MID12 Comp 
                 TAGCTCAGTA CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 51 
                 PGMA MID13 Comp 
                 CACTACTATG CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 52 
                 PGMA MID14 Comp 
                 GTATCTCTCG CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 53 
                 PGMA MID15 Comp 
                 TACGTCGTAT CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
               
                 54 
                 PGMA MID16 Comp 
                 TAGTACGTGA CTGAGTCGGAGACACGCAGGGATGAGATGG 
               
               
                   
               
            
           
           
               
            
               
                 PCR2 Forward Primers 
               
               
                 (Primers with Adapter A, MID, and Forward Tag) 
               
            
           
           
               
               
               
            
               
                 55 
                 PGMA MID1 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGAGTGCGT 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 56 
                 PGMA MID2 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGCTCGACA 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 57 
                 PGMA MID3 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGACGCACTC 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 58 
                 PGMA MID4 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGCACTGTAG 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 59 
                 PGMA MID5 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATCAGACACG 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 60 
                 PGMA MID6 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATATCGCGAG 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 61 
                 PGMA MID7 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGTGTCTCTA 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 62 
                 PGMA MID8 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CTCGCGTGTC 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 63 
                 PGMA MID9 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TAGTATCAGC 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 64 
                 PGMA MID10 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCTCTATGCG 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 65 
                 PGMA MID11 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TGATACGTCT 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 66 
                 PGMA MID12 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TACTGAGCTA 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 67 
                 PGMA MID13 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CATAGTAGTG 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 68 
                 PGMA MID14 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGAGAGATAC 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 69 
                 PGMA MID15 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATACGACGTA 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
               
                 70 
                 PGMA MID16 FT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCACGTACTA 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
            
           
           
               
            
               
                 Primer with Adapter P and Reverse Tag 
               
            
           
           
               
               
               
            
               
                 71 
                 Primer P RT 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
            
           
           
               
            
               
                 PCR2 Reverse Primers 
               
               
                 (Primers with Adapter A, MID, and Reverse Tag) 
               
            
           
           
               
               
               
            
               
                 72 
                 PGMA MID1 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGAGTGCGT 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 73 
                 PGMA MID2 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ACGCTCGACA 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 74 
                 PGMA MID3 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGACGCACTC 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 75 
                 PGMA MID4 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  AGCACTGTAG 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 76 
                 PGMA MID5 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATCAGACACG 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 77 
                 PGMA MID6 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATATCGCGAG 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 78 
                 PGMA MID7 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGTGTCTCTA 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 79 
                 PGMA MID8 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CTCGCGTGTC 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 80 
                 PGMA MID9 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TAGTATCAGC 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 81 
                 PGMA MID10 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCTCTATGCG 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 82 
                 PGMA MID11 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TGATACGTCT 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 83 
                 PGMA MID12 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TACTGAGCTA 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 84 
                 PGMA MID13 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CATAGTAGTG 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 85 
                 PGMA MID14 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  CGAGAGATAC 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 86 
                 PGMA MID15 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  ATACGACGTA 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
               
                 87 
                 PGMA MID16 RT 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG  TCACGTACTA 
               
               
                   
                   
                 TACGGTAGCAGAGACTTGGTCT 
               
               
                   
               
            
           
           
               
            
               
                 Primer with Adapter P and Forward Tag 
               
            
           
           
               
               
               
            
               
                 88 
                 Primer P FT 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
                   
                 ACACTGACGACATGGTTCTACA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 16S Sequences 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID 
                   
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
            
           
           
               
            
               
                 16S V2 Set 2 
               
            
           
           
               
               
               
               
            
               
                  89 
                 16S V2 F101a 
                 GGCGGACGGGTGAGTAA 
                   
               
               
                   
               
               
                  90 
                 16S V2 F101b 
                 GGCGAACGGGTGAGTAA 
                   
               
               
                   
               
               
                  91 
                 16S V2 F101c 
                 GGCGGACGGGTGAGTAA 
                   
               
               
                   
               
               
                  92 
                 16S V2 F101d 
                 GGCGGATGGGTGAGTAA 
                 
                   Lactobacillus 
                 
               
               
                   
               
               
                  93 
                 16S V2 F101e 
                 GGCAAACGGGTGAGTAA 
                 
                   Megasphaera 
                 
               
               
                   
               
               
                  94 
                 16S V2 F101f 
                 GGCGAACGGGCGAGTAA 
                 
                   Mobiluncus 
                 
               
               
                   
               
               
                  95 
                 16S V2 F101g 
                 GGCGAACGGCTGAGTAA 
                 
                   Atopobium 
                 
               
               
                   
               
               
                  96 
                 16S V2 R356a 
                 CACTGCTGCCTCCCGTAG 
                   
               
               
                   
               
               
                  97 
                 16S V2 R356b 
                 TACTGCTGCCTCCCGTAG 
                   
               
               
                   
               
            
           
           
               
            
               
                 16S V3 Set 2 
               
            
           
           
               
               
               
               
            
               
                  98 
                 16S V3 F323a 
                 GACACGGTCCAGACTCCTAC 
                   
               
               
                   
               
               
                  99 
                 16S V3 F323b 
                 GACACGGCCCAGACTCCTAC 
                   
               
               
                   
               
               
                 100 
                 16S V3 F323c 
                 GACACGGTCCAAACTCCTAC 
                 
                   Bacillus 
                 
               
               
                   
               
               
                 101 
                 16S V3 F323d 
                 GACACGGCCCAAACTCCTAC 
                 
                   Lactobacillus 
                 
               
               
                   
               
               
                 102 
                 16S V3 F323e 
                 GATACGGCCCAGACTCCTAC 
                 Myco, Mob,  
               
               
                   
                   
                   
                 Gard 
               
               
                   
               
               
                 103 
                 16S V3 R531a 
                 ATTACCGCGGCTGCTG 
                   
               
               
                   
               
            
           
           
               
            
               
                 PCR1 V2 (Tag, Sequence Specific) 
               
            
           
           
               
               
               
               
            
               
                 104 
                 Tag V2 F101a 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGGACGGGTGAGTAA 
                   
               
               
                   
               
               
                 105 
                 Tag V2 F101b 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGAACGGGTGAGTAA 
                   
               
               
                   
               
               
                 106 
                 Tag V2 F101c 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGCACGGGTGAGTAA 
                   
               
               
                   
               
               
                 107 
                 Tag V2 F101d 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGGATGGGTGAGTAA 
                   
               
               
                   
               
               
                 108 
                 Tag V2 F101e 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCAAACGGGTGAGTAA 
                   
               
               
                   
               
               
                 109 
                 Tag V2 F101f 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGAACGGGCGAGTAA 
                   
               
               
                   
               
               
                 110 
                 Tag V2 F101g 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGGCGAACGGCTGAGTAA 
                   
               
               
                   
               
               
                 111 
                 Tag V2 R356a 
                 TACGGTAGCAGAGACTTGGT 
                   
               
               
                   
                   
                 CTCACTGCTGCCTCCCGTAG 
                   
               
               
                   
               
               
                 112 
                 Tag V2 R356b 
                 TACGGTAGCAGAGACTTGGT 
                   
               
               
                   
                   
                 CTTACTGCTGCCTCCCGTAG 
                   
               
               
                   
               
            
           
           
               
            
               
                 PCR1 V3 (Tag, Sequence Specific) 
               
            
           
           
               
               
               
               
            
               
                 113 
                 Tag V3 F323a 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGACACGGTCCAGACTCCT 
                   
               
               
                   
                   
                 AC 
                   
               
               
                   
               
               
                 114 
                 Tag V3 F323b 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGACACGGCCCAGACTCCT 
                   
               
               
                   
                   
                 AC 
                   
               
               
                   
               
               
                 115 
                 Tag V3 F323c 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGACACGGTCCAAACTCCT 
                   
               
               
                   
                   
                 AC 
                   
               
               
                   
               
               
                 116 
                 Tag V3 F323d 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGACACGGCCCAAACTCCT 
                   
               
               
                   
                   
                 AC 
                   
               
               
                   
               
               
                 117 
                 Tag V3 F323e 
                 ACACTGACGACATGGTTCTA 
                   
               
               
                   
                   
                 CAGATACGGCCCAGACTCCT 
                   
               
               
                   
                   
                 AC 
                   
               
               
                   
               
               
                 118 
                 Tag V3 R531a 
                 TACGGTAGCAGAGACTTGGT 
                   
               
               
                   
                   
                 CTATTACCGCGGCTGCTG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 ITS Sequences 
               
            
           
           
               
               
               
            
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
            
           
           
               
            
               
                 ITS Sequences F and R Primer Pairs 
               
            
           
           
               
               
               
            
               
                 119 
                 ITS1Fb 
                 AAACTCGGTCATTTAGAGGAAGTAA 
               
               
                   
               
               
                 120 
                 ITSR513 
                 GATGCCGGAACCAAGAGAT 
               
               
                   
               
               
                 121 
                 ITSF329 
                 AACCTCCCACCCGTGTTTAT 
               
               
                   
               
               
                 122 
                 ITSR533 
                 ATTTCGCTGCGTTCTTCATC 
               
               
                   
               
               
                 123 
                 ITS1Fb 
                 AAACTCGGTCATTTAGAGGAAGTAA 
               
               
                   
               
               
                 124 
                 ITS2b 
                 GCTGCGTTCTTCATCGATG 
               
               
                   
               
               
                 125 
                 ITSF569 
                 ATCGAGTCTTTGAACGCACA 
               
               
                   
               
               
                 126 
                 ITSR820 
                 CCTACCTGATCCGAGGTCAA 
               
               
                   
               
               
                 127 
                 ITSF570 
                 TCGAGTCTTTGAACGCACAT 
               
               
                   
               
               
                 128 
                 ITSR828 
                 CGGGTATCCCTACCTGATCC 
               
               
                   
               
            
           
           
               
            
               
                 ITS Reading Set (Adapter A, MID or Adapter P) 
               
            
           
           
               
               
               
            
               
                 129 
                 ITSPGM1FbA 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
               
               
                   
                 MID1 
                 ACGAGTGCGTAAACTCGGTCATTTAGAGGA 
               
               
                   
                   
                 AGTAA 
               
               
                   
               
               
                 130 
                 ITSPGMR513P 
                 CCTCTCTATGGGCAGTCGGTGAT  
               
               
                   
                   
                 GATGCCGGAACCAAGAGAT 
               
               
                   
               
               
                 131 
                 TTSPGMF329A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
               
               
                   
                 MID1 
                 ACGAGTGCGTAACCTCCCACCCGTGTTTAT 
               
               
                   
               
               
                 132 
                 ITSPGMR533P 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
                   
                 ATTTCGCTGCGTTCTTCATC 
               
               
                   
               
               
                 133 
                 ITSPGM1FbA 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
               
               
                   
                 MID1 
                 ACGAGTGCGTAAACTCGGTCATTTAGAGGA 
               
               
                   
                   
                 AGTAA 
               
               
                   
               
               
                 134 
                 ITSPGM2bP 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
                   
                 GCTGCGTTCTTCATCGATG 
               
               
                   
               
               
                 135 
                 ITSPGMF569A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
               
               
                   
                 MID1 
                 ACGAGTGCGTATCGAGTCTTTGAACGCACA 
               
               
                   
               
               
                 136 
                 ITSPGMR820P 
                 CCTCTCTATGGGCAGTCGGTGAT  
               
               
                   
                   
                 CCTACCTGATCCGAGGTCAA 
               
               
                   
               
               
                 137 
                 ITSPGMF570A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
               
               
                   
                 MID1 
                 ACGAGTGCGTTCGAGTCTTTGAACGCACAT 
               
               
                   
               
               
                 138 
                 ITSPGMR828P 
                 CCTCTCTATGGGCAGTCGGTGAT  
               
               
                   
                   
                 CGGGTATCCCTACCTGATCC 
               
               
                   
               
            
           
           
               
            
               
                 PCR1 (Tag, Sequence Specific) 
               
            
           
           
               
               
               
            
               
                 335 
                 Tag ITS1Fb 
                 ACACTGACGACATGGTTCTACA 
               
               
                   
                   
                 AAACTCGGTCATTTAGAGGAAGTAA 
               
               
                   
               
               
                 336 
                 Tag ITS2b 
                 TACGGTAGCAGAGACTTGGTCT  
               
               
                   
                   
                 GCTGCGTTCTTCATCGATG 
               
               
                   
               
               
                 337 
                 Tag ITSF569 
                 ACACTGACGACATGGTTCTACA  
               
               
                   
                   
                 ATCGAGTCTTTGAACGCACA 
               
               
                   
               
               
                 338 
                 Tag ITSR820 
                 TACGGTAGCAGAGACTTGGTCT  
               
               
                   
                   
                 CCTACCTGATCCGAGGTCAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   Mycobacterium  Sequences 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID 
                   
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
                 Description 
               
               
                   
               
            
           
           
               
            
               
                   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 
                 MycoPGMF2649A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 GCAAGGTCACCCCGAAG 
                   
               
               
                   
               
               
                 154 
                 MycoPGMR2924P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 CGATGACGCCCTTGTTG 
                   
               
               
                   
               
               
                 155 
                 MycoPGMF2648A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 GGCAAGGTCACCCCGAAGG 
                   
               
               
                   
               
               
                 156 
                 MycoPGMR2934P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 AGGATCTTGCCGATGACG 
                   
               
               
                   
               
               
                 157 
                 MycoPGMF2649A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT GCAAGGTCACCCCGAAG 
                   
               
               
                   
               
            
           
           
               
            
               
                 Fragment 1 Reverse Reading Set (Adapter P or Adapter A) 
               
            
           
           
               
               
               
               
            
               
                 158 
                 MycoPGMF2649P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 GCAAGGTCACCCCGAAG 
                   
               
               
                   
               
               
                 159 
                 MycoPGMR2924A 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
                   
               
               
                   
                   
                 CGATGACGCCCTTGTTG 
                   
               
               
                   
               
               
                 160 
                 MycoPGMF2648P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 GGCAAGGTCACCCCGAAGG 
                   
               
               
                   
               
               
                 161 
                 MycoPGMR2934A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 AGGATCTTGCCGATGACG 
                   
               
               
                   
               
            
           
           
               
            
               
                 Fragment 2 Forward Reading Set (Adapter A or Adapter P) 
               
            
           
           
               
               
               
               
            
               
                 162 
                 MycoPGM2F2898A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 GACGCCACGGCAACAAG 
                   
               
               
                   
               
               
                 163 
                 MycoPGM2F2899A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 ACGCCACGGCAACAAG 
                   
               
               
                   
               
               
                 164 
                 MycoPGM2R3337P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 CAAGTGGTGCAGCTTCAGGATG 
                   
               
               
                   
               
               
                 165 
                 MycoPGM2R3337dP 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 CARGTGGTGCAGCTTCAKGATG 
                   
               
               
                   
               
               
                 166 
                 MycoPGM2R3169P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 GGCGCCGTCGAACAC 
                   
               
               
                   
               
               
                 167 
                 MycoPGM2R3169dP 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 GGCRCCGTCGAACAC 
                   
               
               
                   
               
               
                 168 
                 MycoPGM2F2898A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT GACGCCACGGCAACAAG 
                   
               
               
                   
               
               
                 169 
                 MycoPGM2F2899A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT ACGCCACGGCAACAAG 
                   
               
               
                   
               
            
           
           
               
            
               
                 Fragment 2 Reverse Reading Set (Adapter P or Adapter A) 
               
            
           
           
               
               
               
               
            
               
                 170 
                 MycoPGM2F2898P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 GACGCCACGGCAACAAG 
                   
               
               
                   
               
               
                 171 
                 MycoPGM2F2899P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 ACGCCACGGCAACAAG 
                   
               
               
                   
               
               
                 172 
                 MycoPGM2R3337A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 CAAGTGGTGCAGCTTCAGGATG 
                   
               
               
                   
               
               
                 173 
                 MycoPGM2R3337dA 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 CARGTGGTGCAGCTTCAKGATG 
                   
               
               
                   
               
               
                 174 
                 MycoPGM2R3169A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 GGCGCCGTCGAACAC 
                   
               
               
                   
               
               
                 175 
                 MycoPGM2R3169dA 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 GGCRCCGTCGAACAC 
                   
               
               
                   
               
            
           
           
               
            
               
                 Fragment 3 Forward Reading Set (Adapter A or Adapter P) 
               
            
           
           
               
               
               
               
            
               
                 176 
                 MycoPGM2F3148A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 CACCCCGGTGTTCGAC 
                   
               
               
                   
               
               
                 177 
                 MycoPGM2R3391P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 CTGGGTGATCATCGAGTACG 
                   
               
               
                   
               
               
                 178 
                 MycoPGM2F3148A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT CACCCCGGTGTTCGAC 
                   
               
               
                   
               
            
           
           
               
            
               
                 Fragment 3 Reverse Reading Set (Adapter P or Adapter A) 
               
            
           
           
               
               
               
               
            
               
                 179 
                 MycoPGM2F3148P 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
                   
                 CACCCCGGTGTTCGAC 
                   
               
               
                   
               
               
                 180 
                 MycoPGM2R3391A 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                   
                 CTGGGTGATCATCGAGTACG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                   Streptococcus  Sequences 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID 
                   
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
                 Description 
               
               
                   
               
            
           
           
               
            
               
                   Streptococcus  Fragment 1 
               
            
           
           
               
               
               
               
            
               
                 181 
                 StrepF1475a 
                 CCTTGGGACCTGGTGGTT 
                 saliv therm mitisB 
               
               
                   
                   
                   
                 oralis suis 
               
               
                   
                   
                   
                 anginosus 
               
               
                   
               
               
                 182 
                 StrepF1475b 
                 CCTTAGGACCTGGTGGTT 
                 pyog dysgalac 
               
               
                   
                   
                   
                 canis equi agalac 
               
               
                   
                   
                   
                 pneumo sang 
               
               
                   
               
               
                 183 
                 StrepF1475c 
                 GCTTTAGGTCCTGGTGGTT 
                 mutans 
               
               
                   
               
               
                 184 
                 StrepF1475d 
                 CCTTGGGGCCTGGTGGTT 
                 mitisB 
               
               
                   
               
               
                 185 
                 StrepF1475e 
                 CCTTAGGGCCTGGTGGTT 
                 Parasanguinis 
               
               
                   
               
               
                 186 
                 StrepR1720a 
                 CTTCTTCGTCGGCAGTCAAC 
                 saliv therm pyog 
               
               
                   
                   
                   
                 canis 
               
               
                   
               
               
                 187 
                 StrepR1720b 
                 CTTCTTCATCAGCAGTCAACC 
                 pyog2 agalac 
               
               
                   
                   
                   
                 dysgalac 
               
               
                   
               
               
                 188 
                 StrepR1720c 
                 CTTCTTCATCAGCAGTTAGC 
                 equi 
               
               
                   
               
               
                 189 
                 StrepR1720d 
                 CTTCTTCATCAGCAGTAAGC 
                 mutans 
               
               
                   
               
               
                 190 
                 StrepR1720e 
                 CTTCTTCATCAGCTGTCAAC 
                 pneumo 
               
               
                   
               
               
                 191 
                 StrepR1720f 
                 CTTCTTCATCGGCTGTCAAC 
                 mitis oralis paras 
               
               
                   
                   
                   
                 suis 
               
               
                   
               
               
                 192 
                 StrepR1720g 
                 CTTCCTCGTCAGCGGTCAAC 
                 sang 
               
               
                   
               
               
                 193 
                 StrepR1720h 
                 CTTCTTCGTCCGCTGTCAGC 
                 anginosus 
               
               
                   
               
               
                 194 
                 StrepR1720i 
                 CTTCTTCATCCGCTGTTAGC 
                 intermedius 
               
               
                   
               
            
           
           
               
            
               
                   Streptococcus  Fragment 2 
               
            
           
           
               
               
               
               
            
               
                 195 
                 StrepF1875a 
                 TGCGACAGCATGTATTCCTT 
                   
               
               
                   
               
               
                 196 
                 StrepF1875b 
                 CGCAACAGCATGTATTCCTT 
                 agalac 
               
               
                   
               
               
                 197 
                 StrepF1875c 
                 TGCAACGGCATGTATTCCTT 
                 pyogenes dysgalac 
               
               
                   
                   
                   
                 canis 
               
               
                   
               
               
                 198 
                 StrepF1875d 
                 GGCAACGGCATGTATTCCTT 
                 intermedius 
               
               
                   
               
               
                 199 
                 StrepR2148a 
                 TGAGTTTGAACGACGGAATTT 
                 saliv therm pyog 
               
               
                   
                   
                   
                 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 
                 StrepF2885b 
                 TGAACATTGGTCAGGTTATGG 
                 thermo dysgalac 
               
               
                   
                   
                   
                 sanguin 
               
               
                   
               
               
                 208 
                 StrepF2885c 
                 TGAATATTGGTCAGGTTATGG 
                 pyogenes 
               
               
                   
               
               
                 209 
                 StrepF2885d 
                 TGAATATCGGTCAGGTTATGG 
                 pneumo mitis 
               
               
                   
                   
                   
                 oralis paras 
               
               
                   
               
               
                 210 
                 StrepF2885e 
                 TGAACATCGGACAAGTTATGG 
                 canis 
               
               
                   
               
               
                 211 
                 StrepF2885f 
                 TGAACATTGGACAGGTTATGG 
                 equi 
               
               
                   
               
               
                 212 
                 StrepF2885g 
                 TGAACATTGGGCAAGTTATGG 
                 mutans 
               
               
                   
               
               
                 213 
                 StrepF2885h 
                 TGAATATCGGACAAGTTATGG 
                 agalac intermedius 
               
               
                   
               
               
                 214 
                 StrepF2885i 
                 TGAATATTGGTCAAGTTATGG 
                 anginosus 
               
               
                   
               
               
                 215 
                 StrepR3134a 
                 TGAAGTTTATCATCAACCATGTG 
                 salivarus thermo 
               
               
                   
                   
                   
                 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 
               
               
                   
               
               
                 221 
                 StrepR3134g 
                 TGCAATTTATCGTCAACCATGTG 
                 pneumo 
               
               
                   
               
               
                 222 
                 StrepR3134h 
                 TGGAGCTTATCATCAACCATGTG 
                 anginosus 
               
               
                   
               
            
           
           
               
            
               
                   Streptococcus  Fragment 4 
               
            
           
           
               
               
               
               
            
               
                 223 
                 StrepF3106a 
                 CTTCACCACATGGTTGATGATAA 
                 saliv thermo pyog 
               
               
                   
                   
                   
                 mutans paras suis 
               
               
                   
                   
                   
                 equi 
               
               
                   
               
               
                 224 
                 StrepF3106b 
                 CTCCACCACATGGTTGATGATAA 
                 dysgalac canis 
               
               
                   
                   
                   
                 mitis oralis 
               
               
                   
               
               
                 225 
                 StrepF3106c 
                 CTCCACCACATGGTTGACGATAA 
                 pneumo 
               
               
                   
               
               
                 226 
                 StrepF3106d 
                 CTCCACCACATGGTAGATGATAA 
                 sang 
               
               
                   
               
               
                 227 
                 StrepF3106e 
                 CTTCACCACATGGTAGATGATAA 
                 intermed 
               
               
                   
               
               
                 228 
                 StrepR3366a 
                 TTCTGGTACACCTGGTTTTGG 
                 saliv thermo pyog 
               
               
                   
                   
                   
                 dysgalac paras 
               
               
                   
               
               
                 229 
                 StrepR3366b 
                 TTCTGGCACACCTGGTTTTGG 
                 canis sang 
               
               
                   
               
               
                 230 
                 StrepR3366c 
                 TTCTGGAACACCTGGTTTTGG 
                 agalac pneumo 
               
               
                   
                   
                   
                 mitis oralis suis 
               
               
                   
                   
                   
                 anginosus 
               
               
                   
               
               
                 231 
                 StrepR3366d 
                 TTCTGGGACACCTGGTTTTGG 
                 intermed 
               
               
                   
               
               
                 232 
                 StrepR3366e 
                 TTCTGGTACACCAGGCTTTGG 
                 equi 
               
               
                   
               
               
                 233 
                 StrepR3366f 
                 TTCTGGTACCCCTGGTTTTGG 
                 mutans 
               
               
                   
               
            
           
           
               
            
               
                 PGM Fragment 2 Set (Adapter A, MID or Adapter P) 
               
            
           
           
               
               
               
               
            
               
                 234 
                 StrepPGMF1875a 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 235 
                 StrepPGMF1875b 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 236 
                 StrepPGMF1875c 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 237 
                 StrepPGMF1875d 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 238 
                 StrepPGMR2148a 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 239 
                 StrepPGMR2148b 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 240 
                 StrepPGMR2148c 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 241 
                 StrepPGMR2148d 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 242 
                 StrepPGMR2148e 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 243 
                 StrepPGMR2148f 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 244 
                 StrepPGMR2148g 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
            
           
           
               
            
               
                 PGM Fragment 3 Set (Adapter A, MID or Adapter P) 
               
            
           
           
               
               
               
               
            
               
                 245 
                 StrepPGMF2885a 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 246 
                 StrepPGMF2885b 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 247 
                 StrepPGMF2885c 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 248 
                 StrepPGMF2885d 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 249 
                 StrepPGMF2885e 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 250 
                 StrepPGMF2885f 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 251 
                 StrepPGMF2885g 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 252 
                 StrepPGMF2885h 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 253 
                 StrepPGMF2885i 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 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 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 263 
                 StrepPGMF3106b 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 264 
                 StrepPGMF3106c 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 265 
                 StrepPGMF3106d 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 266 
                 StrepPGMF3106e 
                 CCATCTCATCCCTGCGTGTCTCCGAC TCAG   
                   
               
               
                   
                 MID1 
                 ACGAGTGCGT 
                   
               
               
                   
               
               
                 267 
                 StrepPGMR3366a 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 268 
                 StrepPGMR3366b 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 269 
                 StrepPGMR3366c 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 270 
                 StrepPGMR3366d 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 271 
                 StrepPGMR3366e 
                 CCTCTCTATGGGCAGTCGGTGAT 
                   
               
               
                   
               
               
                 272 
                 StrepPGMR3366f 
                 CCTCTCTATGGGCAGTCGGTGAT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                   Staphylococcus  Sequences 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID 
                   
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
                 Description 
               
               
                   
               
            
           
           
               
            
               
                   Staphylococcus  Fragment 1 
               
            
           
           
               
               
               
               
            
               
                 273 
                 StaphF44a 
                 GAAACTACGCGAGAATTTCAG 
                 aureus, 
               
               
                   
                   
                 AAG 
                 lugdunensis 
               
               
                   
               
               
                 274 
                 StaphF44b 
                 GAAATTACGCGAGAATTTCAG 
                 epidermidis, 
               
               
                   
                   
                 AAG 
                 capitis 
               
               
                   
               
               
                 275 
                 StaphF44c 
                 GAAATTATGCGAGAATTTCAG 
                 haemolyticus 
               
               
                   
                   
                 AAG 
                   
               
               
                   
               
               
                 276 
                 StaphF44d 
                 GAAACTATGCGAGAATTTCAG 
                 saprophyticus 
               
               
                   
                   
                 AGG 
                   
               
               
                   
               
               
                 277 
                 StaphR278a 
                 CGAAGAGGTGCAGCATAAGTA 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 278 
                 StaphR278b 
                 CGTAATGGTGCCGCGTATGTT 
                 intermedius 
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 279 
                 StaphR278c 
                 CGTAGAGGTGCAGAATACGTT 
                 saprophyticus 
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 280 
                 StaphF18a 
                 CCAATATGGAAGACATCGTAA 
                   
               
               
                   
                   
                 ACG 
                   
               
               
                   
               
            
           
           
               
            
               
                   Staphylococcus  Fragment 2 
               
            
           
           
               
               
               
               
            
               
                 281 
                 StaphF1251a 
                 CCAATTCCGTATCGGTTTATC 
                   
               
               
                   
               
               
                 282 
                 StaphF1251b 
                 CCAATTCCGTATTGGTTTATC 
                 lugdunensis, 
               
               
                   
                   
                   
                 saprophyticus 
               
               
                   
               
               
                 283 
                 StaphR1505a 
                 ACTTCCATTTGAGCACGTTC 
                   
               
               
                   
               
               
                 284 
                 StaphR1505b 
                 ACTTCCATTTGGGCACGTTC 
                 caprae 
               
               
                   
               
               
                 285 
                 StaphR1505c 
                 ACTTCCATTTGTGCACGTTC 
                 lugdunensis 
               
               
                   
               
            
           
           
               
            
               
                   Staphylococcus  Fragment 3 
               
            
           
           
               
               
               
               
            
               
                 286 
                 StaphF1484a 
                 GTGAACGTGCTCAAATGGAAG 
                   
               
               
                   
               
               
                 287 
                 StaphF1484b 
                 GTGAACGTGCCCAAATGGAAG 
                 caprae 
               
               
                   
               
               
                 288 
                 StaphF1484c 
                 GTGAACGTGCACAAATGGAAG 
                 lugdunensis 
               
               
                   
               
               
                 289 
                 StaphR1715a 
                 ACATAGCTATCTTCTTCATCA 
                   
               
               
                   
                   
                 GC 
                   
               
               
                   
               
               
                 290 
                 StaphR1715b 
                 ACGTAACTATCCTCTTCATCA 
                 epidermidis 
               
               
                   
                   
                 GC 
                   
               
               
                   
               
               
                 291 
                 StaphR1715c 
                 ACATAGCTATCCTCTTCATCA 
                 epidermidis 
               
               
                   
                   
                 GC 
                   
               
               
                   
               
               
                 292 
                 StaphR1715d 
                 ACATAGCTATCTTCTTCGTCA 
                 aureus 
               
               
                   
                   
                 GC 
                   
               
               
                   
               
               
                 293 
                 StaphR1715e 
                 ACATAACTGTCTTCTTCATCA 
                 lugdunensis 
               
               
                   
                   
                 GC 
                   
               
               
                   
               
            
           
           
               
            
               
                   Staphylococcus  Fragment 4 
               
            
           
           
               
               
               
               
            
               
                 294 
                 StaphF3224a 
                 TCGGTGAGATGGAGGTATGG 
                   
               
               
                   
               
               
                 295 
                 StaphF3224b 
                 TCGGTGAGATGGAAGTATGG 
                 lugdunensis 
               
               
                   
               
               
                 296 
                 StaphF3224c 
                 TCGGTGAAATGGAAGTATGG 
                 saprophyticus 
               
               
                   
               
               
                 297 
                 StaphR3388a 
                 CTCGGAATGATTCTGGAACAC 
                   
               
               
                   
               
               
                 298 
                 StaphR3388b 
                 CTCGGAATGATTCAGGAACAC 
                 intermedius, 
               
               
                   
                   
                   
                 capitis, 
               
               
                   
                   
                   
                 lugdunensis, 
               
               
                   
                   
                   
                 saprophyticus 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                   Burkholderia  Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
               
               
                   
                 ID 
                   
                   
               
               
                   
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
               
                   
                 299 
                 BUR3 
                 GAAGAAGCAGTTCGGCAA 
               
               
                   
               
               
                   
                 300 
                 BUR4 
                 GAGTCGATGACGATCAT 
               
               
                   
               
               
                   
                 301 
                 recAF1 
                 CCACGCTCACGCTGCAGG 
               
               
                   
               
               
                   
                 302 
                 recAR1 
                 CGAGCCCGAGCGCACCAG 
               
               
                   
               
               
                   
                 303 
                 recAF2 
                 CGAAGGCGAGATGGGCG 
               
               
                   
               
               
                   
                 304 
                 recAR2 
                 TCGAGACGCACCGACG 
               
               
                   
               
               
                   
                 305 
                 recAF3 
                 GTGCAGGCGAAGATCGTCG 
               
               
                   
               
               
                   
                 306 
                 recAR3 
                 CCATCGCCTCGGCTTCG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                   Enterococcus  Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
               
               
                   
                 ID 
                   
                   
               
               
                   
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
               
                   
                 307 
                 tufF1 
                 GGCGGACGTCACACTCCATTC 
               
               
                   
               
               
                   
                 308 
                 tufR1 
                 CCGTCTTCGATAGCGATTGGGTGG 
               
               
                   
               
               
                   
                 309 
                 tufF2 
                 GGTTGCTCGTGAAGACATCCAAC 
               
               
                   
               
               
                   
                 310 
                 tufR2 
                 CACCAGTAACGTCTGTTGTACGG 
               
               
                   
               
               
                   
                 311 
                 tufF3 
                 CAGGCGATGATGTTCCAGTTATCGC 
               
               
                   
               
               
                   
                 312 
                 tufR3 
                 GTAGCAACAGTACCACGTCCAGTG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                   Pseudomonas  Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
               
               
                   
                 ID 
                   
                   
               
               
                   
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
               
                   
                 313 
                 APrU 34 
                 TGTAAAACGACGGCCAGTGCNGGR 
               
               
                   
                   
                   
                 TCYTTYTCYTGRCA 
               
               
                   
               
               
                   
                 314 
                 M13 (21) 34 
                 TGTAAAACGACGGCCAGT 
               
               
                   
               
               
                   
                 315 
                 UP1E 34 
                 CAGGAAACAGCTATGACCAYGSNG 
               
               
                   
                   
                   
                 GNGGNAARTTYRA 
               
               
                   
               
               
                   
                 316 
                 M13R 34 
                 CAGGAAACAGCTATGACC 
               
               
                   
               
               
                   
                 317 
                 gyrbF1 
                 CAGCTGGGACATCCTGGCC 
               
               
                   
               
               
                   
                 318 
                 gyrbR1 
                 TGAGGGATGTTGTTGGTAAAGCAC 
               
               
                   
               
               
                   
                 319 
                 gyrbF2 
                 GTGCTTTACCAACAACATCCCTCA 
               
               
                   
               
               
                   
                 320 
                 gyrbR2 
                 TGTCTTTGGTCTGGGAGCTGAAC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 IDT Label Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                   
                   
               
               
                   
                 ID 
                   
                   
               
               
                   
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
               
                   
                 321 
                 Br2-F-MID2 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ACGCTCGACAAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 322 
                 Br2-F-MID3 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 AGACGCACTCAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 323 
                 Br2-F-MID4 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 AGCACTGTAGAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 324 
                 Br2-F-MID5 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ATCAGACACGAGYGGCGIACGGGTGAGTAA 
               
            
           
           
               
               
            
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 325 
                 Br2-F-MID6 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ATATCGCGAGAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 326 
                 Br2-F-MID7 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 CGTGTCTCTAAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 327 
                 Br2-F-MID8 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 CTCGCGTGTCAGYGGCGIACGGGTGAGTAA 
               
               
                   
               
               
                   
                 328 
                 Br3-F-MID2 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ACGCTCGACAACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 329 
                 Br3-F-MID3 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 AGACGCACTCACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 330 
                 Br3-F-MID4 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 AGCACTGTAGACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 331 
                 Br3-F-MID5 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ATCAGACACGACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 332 
                 Br3-F-MID6 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ATATCGCGAGACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 333 
                 Br3-F-MID7 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 CGTGTCTCTAACTCCTACGGGAGGCAGCAG 
               
               
                   
               
               
                   
                 334 
                 Br3-F-MID8 
                 CCATCTCATCCCTGCGTGTCTCCGACTCAG 
               
               
                   
                   
                   
                 ACGAGTGCGTACTCCTACGGGAGGCAGCAG 
               
               
                   
               
            
           
         
       
     
     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 
     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. 
     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. 
     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. 
     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 
     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. 
     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. 
     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. 
     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. 
     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 
     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. 
     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. 
     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. 
     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 
     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. 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Reagents 
                 x1 (uL) 
                 x30 (uL) 
                 x60 (uL) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 16Sv3v4-F Primer (1 uM) 
                 5 
                 150 
                 300 
               
               
                   
                 16Sv3v4-R Primer (1 uM) 
                 5 
                 150 
                 300 
               
               
                   
                 2X KAPA HiFi HotStart 
                 12.5 
                 375 
                 750 
               
               
                   
                 Ready Mix 
                   
                   
                   
               
               
                   
                 Total 
                 22.5 
                 675 
                 1,350 
               
               
                   
                   
               
            
           
         
       
     
     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. 
     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. 
     Results 
     Sequence Metrics 
     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 &gt;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). 
     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 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 % of  
                 reads 
                   
               
               
                   
                   
                 PF 
                 clustered 
                 OTUs 
               
               
                 Sample 
                 Description 
                 reads 
                 in OTUs 
                 (&gt;0.5%) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 M1 
                 mixed organisms 
                 4.37 
                 13681 
                 7 
               
               
                 M2 
                 mixed organisms 
                 4.05 
                 11764 
                 14 
               
               
                 M3 
                 mixed organisms 
                 6.71 
                 19967 
                 8 
               
               
                 M4 
                 mixed organisms 
                 3.65 
                 10087 
                 11 
               
               
                 M5 
                 mixed organisms 
                 7.77 
                 19821 
                 8 
               
               
                 M6 
                 mixed organisms 
                 3.30 
                 7232 
                 6 
               
               
                 S1 
                 pure sample 
                 5.55 
                 19673 
                 1 
               
               
                 S2 
                 pure sample 
                 2.51 
                 8874 
                 1 
               
               
                 S3 
                 pure sample 
                 4.39 
                 15577 
                 2 
               
               
                 S4 
                 pure sample 
                 2.27 
                 8073 
                 1 
               
               
                 S5 
                 pure sample 
                 6.78 
                 23824 
                 1 
               
               
                 S6 
                 pure sample 
                 3.05 
                 10899 
                 1 
               
               
                 P1 
                 patient sample 
                 2.78 
                 9731 
                 1 
               
               
                 P2 
                 patient sample 
                 1.46 
                 5147 
                 1 
               
               
                 P3 
                 patient sample 
                 2.21 
                 7705 
                 1 
               
               
                 NEG 
                 Negative Ctrl 
                 0.007 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     Organism Identification is shown in Table 12 below: 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Identification of Pure (S1-S6) and Patient (P1-P3) Samples 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 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 
                   
               
               
                 S3_S9 
                   Enterobacter _cloacae 
                 425 
                 99.5% 
                 31.0% 
                 4824 
                 
                   Salmonella cholereasuis 
                 
               
               
                 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 
                 
               
               
                   
               
            
           
         
       
     
     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. 
     Table 13 below demonstrates successful recovery of most input species from the mixed samples.  E. cloacae , used in samples M1 and M2, appeared as a contaminant in samples M3 and M5. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Identification of Six Mixed Bacterial Samples 
               
            
           
           
               
               
               
               
               
               
            
               
                 Sample 
                 OTUId 
                 Reads 
                 Pct 
                 Identity 
                 Input 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M1_S1 
                 OTU_1 
                 5580 
                  40.8% 
                 
                   Acinetobacter baumannii 
                 
                 
                   Acinetobacter baumannii 
                 
                   
               
               
                 M1_S1 
                 OTU_2 
                 2480 
                  18.1% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Escherichia coli 
                 
                 
                   Shigella sonnei 
                 
               
               
                 M1_S1 
                 OTU_3 
                 2047 
                  15.0% 
                 
                   Enterobacter aerogenes 
                 
                 
                   Enterobacter aerogenes 
                 
                   
               
               
                 M1_S1 
                 OTU_5 
                 1614 
                  11.8% 
                 
                   Enterobacter cloacae 
                 
                 
                   Enterobacter cloacae 
                 
                   
               
               
                 M1_S1 
                 OTU_4 
                 861 
                  6.3% 
                 
                   Bordetella pertussis 
                 
                 
                   Bordetella pertussis 
                 
                   
               
               
                 M1_S1 
                 OTU_6 
                 573 
                  4.2% 
                 
                   Salmonella enterica 
                 
                 
                   Salmonella choleraesuis 
                 
                   
               
               
                 M1_S1 
                 OTU_7 
                 518 
                  3.8% 
                 
                   Klebsiella variicola 
                 
                 
                   Klebsiella pneumoniae 
                 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   Nocardia farcinica 
                 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M2_S2 
                 OTU_1 
                 3984 
                  33.9% 
                 
                   Staphylococcus epide rmidis/capitis  
                 
                 
                   Staphylococcus epidermidis 
                 
                 
                   Staphylococcus aureus 
                 
               
               
                 M2_S2 
                 OTU_15 
                 1121 
                  9.5% 
                 
                   Staphylococcus saprophyticus 
                 
                 
                   Staphylococcus saprophyticus 
                 
                   
               
               
                 M2_S2 
                 OTU_3 
                 1058 
                  9.0% 
                 
                   Streptococcus pyogenes 
                 
                   Strep pyogenes  group a 
                   
               
               
                 M2_S2 
                 OTU_2 
                 1032 
                  8.8% 
                 
                   Acinetobacter baumannii 
                 
                 
                   Acinetobacter baumannii 
                 
                   
               
               
                 M2_S2 
                 OTU_5 
                 934 
                  7.9% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Shigella sonnei 
                 
                 
                   Escherichia coli 
                 
               
               
                 M2_S2 
                 OTU_6 
                 807 
                  6.9% 
                 
                   Streptococcus agalactiae 
                 
                   Strep agalactiae  group b 
                   
               
               
                 M2_S2 
                 OTU_4 
                 742 
                  6.3% 
                   Neisseria _lactamica 
                 
                   Neisseria lactamica 
                 
                   
               
               
                 M2_S2 
                 OTU_7 
                 574 
                  4.9% 
                 
                   Proteus mirabilis 
                 
                 
                   Proteus mirabilis 
                 
                   
               
               
                 M2_S2 
                 OTU_8 
                 416 
                  3.5% 
                 
                   Enterobacter aerogenes 
                 
                 
                   Enterobacter aerogenes 
                 
                   
               
               
                 M2_S2 
                 OTU_9 
                 296 
                  2.5% 
                 
                   Enterobacter cloacae 
                 
                 
                   Enterobacter cloacae 
                 
                   
               
               
                 M2_S2 
                 OTU_11 
                 274 
                  2.3% 
                 
                   Haemophilus aegyptius 
                 
                 
                   Haemophilus influenzae 
                 
                   
               
               
                 M2_S2 
                 OTU_12 
                 179 
                  1.5% 
                 
                   Bordetella pertussis 
                 
                 
                   Bordetella parapertussis 
                 
                 
                   Bordetella pertussis 
                 
               
               
                 M2_S2 
                 OTU_10 
                 166 
                  1.4% 
                 
                   Pseudomonas aeruginosa 
                 
                 
                   Pseudomonas aeruginosa 
                 
                   
               
               
                 M2_S2 
                 OTU_13 
                 108 
                  0.9% 
                 
                   Salmonella enterica 
                 
                 
                   Salmonella choleraesuis 
                 
                   
               
               
                 M2_S2 
                 OTU_14 
                 73 
                  0.6% 
                   Klebsiella _variicola 
                 
                   Klebsiella pneumoniae 
                 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   Ochrobactrum anthropi 
                 
                   
                   
               
               
                   
                   
                   
                 
                   Camphylobacter jejuni 
                 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M3_S3 
                 OTU_1 
                 7103 
                  35.6% 
                 
                   Staphylococcus capitis 
                 
                 
                   Staphylococcus aureus 
                 
                   
               
               
                   
                 OTU_6 
                 4117 
                  20.6% 
                 
                   Staphylococcus saprophyticus 
                 
                 
                   Staphylococcus saprophyticus 
                 
                   
               
               
                   
                 OTU_2 
                 3380 
                  16.9% 
                   Proteus _mirabilis 
                 
                   Proteus mirabilis 
                 
                   
               
               
                   
                 OTU_3 
                 2082 
                  10.4% 
                 
                   Streptococcus agalactiae 
                 
                   Strep agalactiae  group b 
                   
               
               
                   
                 OTU_4 
                 1396 
                  7.0% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Escherichia coli 
                 
                   
               
               
                   
                 OTU_5 
                 1046 
                  5.2% 
                 
                   Pseudomonas aeruginosa 
                 
                 
                   Pseudomonas aeruginosa 
                 
                   
               
               
                   
                 OTU_7 
                 574 
                  2.9% 
                 
                   Salmonella enterica 
                 
                 
                   Salmonella choleraesuis 
                 
                   
               
               
                   
                 OTU_8 
                 269 
                  1.3% 
                   Enterobacter _cloacae 
                   
                   
               
               
                 M4_S4 
                 OTU_1 
                 3999 
                  39.6% 
                 
                   Staphylococcus epidermidis/capitis  
                 
                 
                   Staphylococcus epidermidis 
                 
                   
               
               
                 M4_S4 
                 OTU_2 
                 1677 
                  16.6% 
                 
                   Streptococcus pyogenes 
                 
                   Strep pyogenes  group a 
                   
               
               
                 M4_S4 
                 OTU_3 
                 1215 
                  12.0% 
                 
                   Acinetobacter baumannii 
                 
                 
                   Acinetobacter baumannii 
                 
                   
               
               
                 M4_S4 
                 OTU_4 
                 1069 
                  10.6% 
                 
                   Neisseriajactamica 
                 
                 
                   Neisseria lactamica 
                 
                   
               
               
                 M4_S4 
                 OTU_5 
                 533 
                  5.3% 
                 
                   Enterobacter aerogenes 
                 
                 
                   Enterobacter aerogenes 
                 
                   
               
               
                 M4_S4 
                 OTU_6 
                 490 
                  4.9% 
                 
                   Haemophilus aegyptius 
                 
                 
                   Haemophilus influenzae 
                 
                   
               
               
                 M4_S4 
                 OTU_7 
                 369 
                  3.7% 
                 
                   Enterobacter cloacae 
                 
                 
                   Enterobacter cloacae 
                 
                   
               
               
                 M4_S4 
                 OTU_8 
                 240 
                  2.4% 
                   Bordetella _pertussis 
                 
                   Bordetella parapertussis 
                 
                 
                   Bordetella pertussis 
                 
               
               
                 M4_S4 
                 OTU_10 
                 222 
                  2.2% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Shigella sonnei 
                 
                   
               
               
                 M4_S4 
                 OTU_9 
                 137 
                  1.4% 
                 
                   Salmonella enterica 
                 
                 
                   Salmonella choleraesuis 
                 
                   
               
               
                 M4_54 
                 OTU_11 
                 132 
                  1.3% 
                 
                   Klebsiella variicola 
                 
                 
                   Klebsiella pneumoniae 
                 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   Nocardia farcinica 
                 
                   
                   
               
               
                   
                   
                   
                 
                   Ochrobactrum anthropi 
                 
                   
                   
               
               
                   
                   
                   
                 
                   Camphylobacter jejuni 
                 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M5_S5 
                 OTU_1 
                 6110 
                 437.7% 
                 
                   Streptococcus pyogenes 
                 
                   Strep pyogenes  group a 
                   
               
               
                 M5_S5 
                 OTU_2 
                 4336 
                 310.6% 
                 
                   Acinetobacter baumannii 
                 
                 
                   Acinetobacter baumannii 
                 
                   
               
               
                 M5_S5 
                 OTU_3 
                 3311 
                 237.2% 
                 
                   Neisseria lactamica 
                 
                 
                   Neisseria lactamica 
                 
                   
               
               
                 M5_S5 
                 OTU_4 
                 3151 
                 225.7% 
                 
                   Proteus mirabilis 
                 
                 
                   Proteus mirabilis 
                 
                   
               
               
                 M5_S5 
                 OTU_5 
                 1526 
                 109.3% 
                   Haemophilus _aegyptius 
                 
                   Haemophilus influenzae 
                 
                   
               
               
                 M5_S5 
                 OTU_6 
                 720 
                  51.6% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Shigella sonnei 
                 
                   
               
               
                 M5_S5 
                 OTU_7 
                 453 
                  32.4% 
                   Salmonella _enterica 
                 
                   Salmonella choleraesuis 
                 
                   
               
               
                 M5_S5 
                 OTU_8 
                 207 
                  14.8% 
                   Enterobacter _cloacae 
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   Nocardia farcinica 
                 
                   
                   
               
               
                   
                   
                   
                 
                   Corynebacterium 
                 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 M6_S6 
                 OTU_3 
                 1697 
                  23.5% 
                   Enterobacter _aerogenes 
                 
                   Enterobacter aerogenes 
                 
                   
               
               
                 M6_S6 
                 OTU_4 
                 1546 
                  21.4% 
                   Bordetella _pertussis 
                 
                   Bordetella pertussis 
                 
                 
                   Bordetella parapertussis 
                 
               
               
                 M6_S6 
                 OTU_5 
                 1206 
                  16.7% 
                 
                   Enterobacter cloacae 
                 
                 
                   Enterobacter cloacae 
                 
                   
               
               
                 M6_S6 
                 OTU_2 
                 1204 
                  16.6% 
                 
                   E. fergusonii/E. coli/Shigella 
                 
                 
                   Escherichia coli 
                 
                   
               
               
                 M6_S6 
                 OTU_1 
                 1131 
                  15.6% 
                 
                   Pseudomonas aeruginosa 
                 
                 
                   Pseudomonas aeruginosa 
                 
                   
               
               
                 M6_S6 
                 OTU_6 
                 448 
                  6.2% 
                 
                   Klebsiella variicola 
                 
                 
                   Klebsiella pneumoniae 
                 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 
                   Camphylobacter jejuni 
                 
                   
                   
               
               
                   
               
            
           
         
       
     
     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 
     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. 
     Results 
     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.