Reagents, methods, and kits for identifying organisms across taxonomic domains

Abstract

Described herein are reagents, methods, and kits for identifying organisms across multiple taxonomic domains in a sample. The method includes performing a multiplex polymerase chain reaction (PCR) with the sample using a plurality of primer pairs that are capable of amplifying at least one identifying nucleic acid of organisms across multiple taxonomic domains, thereby generating amplicons of organisms present in the sample. The method further includes performing sequencing on the amplicons to obtain sequencing reads of the amplicons. The method further includes identifying organisms in the sample based on the sequencing reads. Also described herein are kits for perfoming the methods contemplated herein.

Description

REAGENTS, METHODS, AND KITS FOR IDENTIFYING ORGANISMS ACROSSTAXONOMIC DOMAINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 540,770, filed September 27, 2023, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The XML file named "046528-7124W01(01079)_Seq Listing.XML" created on September 24, 2024, comprising 18,124 bytes, is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Microbiomes colonize almost every part of the world, as well as environments within the body or on the surface of the body of animals, such as humans. Microbiomes are often polydomain in nature (z.e., includes organisms from more than one taxonomic domain) and include bacteria as well as eukaryotic organisms such as fungi, alveolates - including apicomplexan parasites, trypanosomes, algae, and other protozoa and small protists.

[0004] Accurate identification of organisms in microbiomes, such as identification down to the species level or strain level, is important for the studies of ecology, host-symbiont systems, and diseases caused by or involving microorganisms and dysbiosis of microbiomes. However, effective tools for identifying an organism in a polydomain microbiome are lacking, and there is a need to develop tools that can efficiently and reproducibly identify large numbers, such as hundreds or thousands of species, in a polydomain microbiome. The present invention addresses this need.SUMMARY

[0005] In some aspects, the present invention is directed to the following non-limiting embodiments:Method of identifying microorganisms across multiple taxonomic domains

[0006] In some aspects, the present invention is directed to a method of identifying microorganisms across multiple taxonomic domains in a sample.

[0007] In some embodiments, the method comprises: amplifying one or more identifying nucleic acids of a plurality of microorganisms across multiple taxonomic domains in the sample with a multiplex polymerase chain reaction (PCR) using a plurality of primer pairs, thereby generating amplicons of a plurality of microorganisms present in the sample; performing sequencing on the amplicons to obtain sequencing reads of the amplicons; and identifying the plurality of microorganisms in the sample based on the sequencing reads,

[0008] In some embodiments, a first identifying nucleic acid present in a first group of microorganisms from a first taxonomic domain is amplified by a first primer pair of the plurality of primer pairs.

[0009] In some embodiments, the first primer pair binds to two conserved regions at two ends of the at least one first identifying nucleic acid shared by the microorganisms in the first group.

[0010] In some embodiments, a second identifying nucleic acid present in a second group of microorganisms from a second taxonomic domain is amplified by a second primer pair of the plurality of primer pairs.

[0011] In some embodiments, the second primer pair binds to two conserved regions at two ends of the at least one second identifying nucleic acid shared by the microorganisms in the second group.

[0012] In some embodiments, the sample is at least one selected from a metazoan sample (such as an animal host sample or a vector sample), a plant sample (such as a plant host sample or an agricultural sample), and an environmental sample (such as a sample from an industrial site or the nature).

[0013] In some embodiments, the multiple taxonomic domains comprise a prokaryotic domain and Eukaryotes.

[0014] In some embodiments, the method further comprises attaching a first barcode sequence to the amplicon from the microorganisms from the first group, and attaching a second barcode sequence to the amplicon from the microorganisms from the second group.

[0015] In some embodiments, the plurality of primer pairs does not amplify any identifying nucleic acid in the host.

[0016] In some embodiments, the method further comprises removing a nucleic acid from a non-microbial source amplifiable by two or more primers of the plurality of primer pairs from the sample.

[0017] In some embodiments, the nucleic acid from a non-microbial source is a nucleic acid from a metazoan or plant species serving as the host or the vector of the microorganisms.

[0018] In some embodiments, the nucleic acid from a non-microbial is removed by a gene editing method.

[0019] In some embodiments, the group of microorganisms comprises organisms in a taxonomic domain, a kingdom, a phylum, a class, an order, or a family.

[0020] In some embodiments, the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the bacterial domain and at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the eukaryal domain.

[0021] In some embodiments, the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of bacteria, at least one primer pair amplifying at least one identifying nucleic acid of fungi, or at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

[0022] In some embodiments, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Amoebozoa parasite, and an Alveolate parasite.

[0023] In some embodiments, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acids of bacteria. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence set forth in SEQ ID NO:1 and a second primer comprising the sequence set forth in SEQ ID NO: 1. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence setforth in SEQ ID N0:5 and a second primer comprising the sequence set forth in SEQ ID NO:6. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO:8.

[0024] In some embodiments, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of fungi. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence set forth in SEQ ID NO:9 and a second primer comprising the sequence set forth in SEQ ID NO: 10.

[0025] In some embodiments, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites. In some embodiments, the at least one primer pair comprises a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO:12.

[0026] In some embodiments, the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

[0027] In some embodiments, the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene of species / strains in the eukarya domain, ITS1 rRNA sequences, 5.8S genes, ITS2 RNA sequence and 28S rRNA genes, or any combinations thereof.

[0028] In some embodiments, the plurality of primer pairs are configured that the amplicons of the plurality of microorganisms generated by the multiplex PCR has a minimal length of 1,300 base pairs.

[0029] In some embodiments, the host is a mammal. In some embodiments, the host is a human.

[0030] In some embodiments, the sample obtained from the host comprises at least one selected from the group consisting of a blood sample, a bronchial alveolar lavage specimen, a bile duct specimen, a cerebrospinal fluid (CSF) sample, a dental sample, a fecal sample, a gastrointestinal aspirate specimen, a medical implant specimen, an indwelling pump specimen, a middle-ear effusion specimen, a mucosal sample, an ocular specimen, a plasma specimen, periodontal crevicular specimen, a saliva sample, a serum specimen, a skin swab specimen, a surgical debridement specimen, a synovial fluid sample, a tissue biopsy sample, a urine sample, and a wound specimen.

[0031] In some embodiments, the dental sample comprises at least one selected from the group consisting of a caries specimen, a dental implant specimen, a dental plaque specimen, an endodontic pulp specimen, a periodontal crevicular specimen, a periodontal tissue biopsy specimen, and a salivary specimen.

[0032] In some embodiments, the vector is an arthropod vector of infectious disease, optionally an arachnid sample or an insect sample.

[0033] In some embodiments, the arachnid sample comprises or is derived from at least one selected from the group consisting of a tick, a spider, a mite, and a chigger.

[0034] In some embodiments, the insect sample comprises or is derived from at least one selected from the group consisting of a mosquito, a biting fly, a flea, a louse, a bed bug, a bat bug, and a sand fly.

[0035] In some embodiments, the sample is an environment sample, a veterinary sample, an agricultural sample, an aquaculture sample, an industrial sample, or combinations thereof.

[0036] In some embodiments, performing sequencing on the amplicons of the sample comprises performing circular consensus sequencing on the amplicons.

[0037] In some embodiments, identifying microorganisms in the sample based on results of the sequencing comprises comparing sequencing reads obtained in the sequencing step or processed sequencing results thereof with a nucleic acid sequence database.

[0038] In some embodiments, identifying microorganisms in the sample based on results of the sequencing comprises performing sequence alignment on the sequencing reads or processed sequencing results thereof against known nucleic acid sequences of microorganisms.Kit

[0039] In some aspects, the present invention is directed to a kit.

[0040] In some embodiments, the kit comprises a first primer pair for amplifying at least one identifying nucleic acid in a first group of microorganisms; and a second primer pair for amplifying at least one identifying nucleic acid in a second group or microorganisms.

[0041] In some embodiments, the first group of microorganisms and the second group of microorganisms are from different taxonomic domains.

[0042] In some embodiments, the first primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the first group of microorganisms.

[0043] In some embodiments, the second primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the second group of microorganisms.

[0044] In some embodiments, each primer in the first primer pair and the second primer pair comprises a barcoding sequence unique to the primer pair the primer belongs to.

[0045] In some embodiments, the first primer pair and the second primer pair are configured such that no identifying nucleic acid from a host of the first group of microorganisms or the second group of microorganisms are amplified by the primer pairs.

[0046] In some embodiments, the kit further comprises a first barcode for attaching to amplicons generated by the first primer pair and a second barcode for attaching to amplicons generated by the second primer pair.

[0047] In some embodiments, the first group or the second group comprises microorganisms in a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.

[0048] In some embodiments, the first primer pair amplifies at least one identifying nucleic acid of microorganisms in the bacteria domain and the second primer pair amplifies at least one identifying nucleic acid of microorganisms in the eukarya domain.

[0049] In some embodiments, the first primer pair and the second primer pair comprise at least two selected from the group consisting of a primer pair amplifying at least one identifying nucleic acid of bacteria, a primer pair amplifying at least one identifying nucleic acid of fungi, a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

[0050] In some embodiments, at least one of the first primer pair or the second primer pair amplifies at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Aloebozoa parasite, and an Alveolate parasite.

[0051] In some embodiments, the first primer pair and the second primer comprises at least two selected from the group consisting of(a) a primer pair amplifying at least one identifying nucleic acid of bacteria, comprising: a first primer comprising the sequence set forth in SEQ ID NO: 1 and a second primer comprising the sequence set forth in SEQ ID NO: 1, a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4, a first primer comprising the sequence set forth in SEQ ID NO: 5 and a second primer comprising the sequence set forth in SEQ ID NO:6, or afirst primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO:8;(b) a primer pair amplifying at least one identifying nucleic acid of fungi, comprising a first primer comprising the sequence set forth in SEQ ID NO:9 and a second primer comprising the sequence set forth in SEQ ID NO: 10; and(c) a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, comprising a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO: 12.

[0052] In some embodiments, the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

[0053] In some embodiments, the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene, a ITS1 rRNA sequence, a 5.8S gene, an ITS2 RNA sequence, a 28S rRNA gene of species / strains in the eukarya domain, or combinations thereof.

[0054] In some embodiments, an amplicon amplified by the first primer pair or the second primer pair has a length of 1,250 base pair or longer.

[0055] In some embodiments, the kit further comprises a polynucleic acid adaptor for converting an amplicon of a PCR to a circular DNA for circular consensus sequencing.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, nonlimiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0057] Figs. 1 A-1C illustrate certain aspects of a method of identifying organism across multiple taxonomic domains in a sample, in accordance with some embodiments. Fig. 1 A: Illustration of a non-limiting sample. Fig. IB: Illustration of a non-limiting multiplex polymerase chain reaction (PCR). Fig. 1C: Illustration of a non-limiting process of identifying species / strains from sequencing reads.

[0058] Fig. 2 illustrates the attachment of barcode sequences to amplicons, in accordance with some embodiments.DETAILED DESCRIPTION

[0059] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0060] In one aspect, the study described herein has identified tools for characterizing a large number of species and strains of organisms across multiple taxonomic domains in a sample of comprising a polymicrobial assemblage. In certain embodiments, the methods of the disclosure do not require multiple tests for identifying organisms in each taxonomic domain.

[0061] Accordingly, in some aspects, the present disclosure is directed to a method of identifying organisms across multiple taxonomic domains in a sample.

[0062] In some aspects, the present disclosure is directed to a kit for identifying organisms across multiple taxonomic domains in a sample.Definitions

[0063] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, biochemistry, and microbiology are those well- known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to beinterpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0064] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0065] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0066] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0067] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.Method of Identifying Organisms Across Multiple Taxonomic Domains in Sample

[0068] In some aspects, the present disclosure is directed to a method of identifying organisms across multiple taxonomic domains in a sample.

[0069] In some embodiments, the organisms are microorganisms. In some embodiments, the organisms are microorganismal pathogens, such as pathogens that affects plants, animals (both vertebrates and invertebrates), and mammals, such as humans.

[0070] In some embodiments, the method includes performing a multiplex polymerase chain reaction (PCR) with the sample using a plurality of primer pairs capable of amplifying at leastone identifying nucleic acid of a plurality of organisms across multiple taxonomic domains, thereby generating amplicons of the plurality of organisms present in the sample.

[0071] In some embodiments, the method includes performing sequencing (e.g., a nucleic acid sequencing such as DNA sequencing or RNA sequencing) on the amplicons to obtain sequencing reads of the amplicons.

[0072] In some embodiments, the method includes identifying the plurality of organisms in the sample based on the sequencing reads.

[0073] In some embodiments, the at least one identifying nucleic acid amplifiable by a primer pair of the plurality of primer pairs exists in a group of organisms. In some embodiments, the primer pair binds to two conserved regions at two ends of the at least one identifying nucleic acid shared by the organisms in the group.

[0074] In some embodiments, the method further comprising attaching barcode sequences to the amplicons, such as by performing a second PCR step using a second primer pair comprising the barcode sequences.

[0075] In some embodiments, the group of organisms includes organisms in a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.Sample

[0076] Considering that polymicrobial assemblage (such as microbiomes) comprising microorganisms across multiple taxonomic domains can be found in virtually any place on earth, all the way from underground spaces to the atmosphere, and can locate within life forms such as animals and plants, the source of the sample is not limited.

[0077] In some embodiments, the sample is from a host of a polymicrobial assemblage, such as a metazoan (animal) host or a plant host. Non-limiting examples of host samples include patient samples, a veterinary samples, agricultural samples, and the like.

[0078] In some embodiments, the sample is from a vector that transmit the polymicrobial assemblage into or between hosts. Non-limiting examples of such samples include arthropod samples such as mosquito samples, tick samples, Triatominae samples, flea samples, and the like.

[0079] In some embodiments, the sample is from the environment, such a soil, water, air, rock, or the like. Non-limiting examples of environmental samples include a sample from the nature, an industrial sample, and the like.

[0080] In some embodiments, the sample is from the host or the vector. In some embodiments, the sample includes a polymicrobial assemblage that colonizes or parasitizes particular environments within the body and / or on the surface of the body of the host or the vector.

[0081] In some embodiments, the polymicrobial assemblage colonizes the skin, oral cavity, gastrointestinal tract, respiratory tract, urogenital tract, a wound, or the like, of a subject, and the sample is drawn from one or more of these locations.

[0082] In some embodiments, the sample includes a polymicrobial assemblage that colonizes a soil, air, a body or water, or any other terrestrial, aquatic, and / or atmospheric environments.

[0083] In some embodiments, the microbiome includes a poly domain community (z.e., includes organisms from more than one taxonomic domain). In some embodiments, the microbiome includes bacteria, fungi, and / or protozoa.

[0084] In some embodiments, the sample is collected from a subject (such as a mammal, such as a human) suspected or confirmed to be suffering from a disease or disorder caused by or involving one or more polydomain microbiomes. Examples of diseases or disorders caused by or involving polydomain microbiomes include adenoiditis, bacterial vaginosis, burn wounds, chronic and recurrent otitis media, chronic infections, chronic obstructive pulmonary disease, cystic fibrosis lung disease, diabetic ulcers, Lyme disease, periprosthetic joint infections, pressure sores, salpingitis, sinusitis, surgical wounds, tonsilitis, venous stasis ulcers. Examples of diseases or disorders caused by or involving polydomain microbiomes further includes biofilms associated with any foreign body implant, which includes arthro-prostheses, catheters (such as vascular, urinary ventriculoperitoneal shunts), breast implants, cochlear implants, coronary stents, dental implants, fabric, fracture-fixation devices, heart valves, inflatable penile implants, implantable neurological stimulators, intra-ocular lenses, meshes, pace-makers, plates, sutures, screws, ventricular assist devices, and the like. One of ordinary skill in the art would understand that the lists in this paragraph are far from comprehensive as due to the lack of methods of identifying organisms in polydomain microbiomes such as the instant methods, many diseasesand disorders are being studied and / or treated mistakenly as caused by single domain microbiomes or even single species.

[0085] In some embodiments, the sample is a biological sample from a subject, such as a blood sample, a bronchial alveolar lavage specimen, a bile duct specimen, a cerebrospinal fluid (CSF) sample, a fecal sample, a gastrointestinal aspirate specimen, a medical implant specimen (such as a specimen from an artificial joint, a spinal instrumentation, a fixation hardware for orthopedic fractures, a pacemaker, an indwelling catheter such as a urinary or circulatory catheter, a CSF ventriculoperitoneal shunt, and the like), an indwelling pump specimen (such as a specimen from an insulin pump, or a drug delivery pump), a middle-ear effusion specimen, a mucosal sample (such as an oral, nasopharyngeal, oropharyngeal, bronchial, or gastrointestinal specimen), an ocular (tear) specimen, a plasma specimen, periodontal crevicular specimen, a saliva sample, a serum specimen, a skin swab specimen, a surgical debridement specimen, a synovial fluid sample, a tissue biopsy sample, a urine sample, a wound specimen, or the like.

[0086] In some embodiments, the sample is a dental sample from a subject. Examples of dental samples include a caries specimen, a dental implant specimen, a dental plaque specimen, an endodontic pulp specimen, a periodontal crevicular specimen, a periodontal tissue biopsy specimen, a salivary specimen, and the like.

[0087] In some embodiments, the subject is a mammal, such as a bovine animal, a canine animal, an equus animal, an even-toed ungulate animal, a feline animal, a primate animal, or the like. In some embodiments, the subject is a human.

[0088] In some embodiments, the subject is a bird, such as chicken, a turkey, a duck, a pheasant.

[0089] In some embodiments, the sample is or is derived from an arthropod vector of infectious disease. Examples of arthropod vectors of infectious diseases include arachnids such as ticks, spiders, mites, chiggers, and the like; and insects such as mosquitos, biting flies, fleas, lice, bed bugs, bat bugs, sand flies, and the like.

[0090] In some embodiments, the sample is a sample collected from the environment. Examples of samples collected from the environment include an agricultural sample, a plant sample, a rock sample, a soil sample, a sewage sample, a water sample, and the like. In some embodiments, applying the method or kits to plant samples allows the study of mutualistic, commensal, saprophytic, parasitic, and pathogenic microbiomes of plants.

[0091] In some embodiments, organisms or microorganisms of interest that are potentially present in the sample are organized into several groups, each group includes identifying nucleic acids, which are suitable for the identification of the organism and amplifiable by a primer pair. The identifying nucleic acids in the same group share conserved regions at two ends thereof, and the primer pair bind to the two conserved regions. In some embodiments, each group is a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof. One of ordinary skill in the art would understand that organisms within the same taxonomic group often share related nucleic acid sequences that include conserved regions, which can be used as primer binding sites for PCR amplification.

[0092] Fig. 1A illustrates certain aspects of the sample, in accordance with some embodiments.

[0093] Referring to Fig. 1A, in some embodiments, the sample 10 includes a first organism 110, a second organism 120, a third organism 210, and a fourth organism 220. It is worth noting that the sample can include significantly more types of organisms than just four, and the limited number of organisms shown in Fig. 1A is only for the purpose of simplifying the illustration. In some embodiments, the same include 10 types or more of organisms, such as 20 types or more, 50 types or more, 100 types or more, 200 types or more, 500 types or more, 1,000 types or more, 2,000 types or more, or 5,000 types or more of organisms.

[0094] Referring to Fig. 1 A, in some embodiments the first organism 110 and the second organism 120 belong to a first taxonomic domain, and the third organism 210 and the fourth organism 220 belong to a second taxonomic domain different from the first taxonomic domain. In some embodiments, the first domain and the second domain are both independently the bacteria domain, or the eukarya domain, as long as the first domain and the second domain are different.

[0095] In some embodiments, the first organism 110 include a first nucleic acid 111, and the second organism include a second nucleic acid 121. The first organism and the second organism are of different species or strains and have different sequences between the first nucleic acid 111 and the second nucleic acid 121. The first nucleic acid 111 and the second nucleic acid 121, however; both include the conserved regions 101 and 103. In some embodiments, the first nucleic acid 111 and the second nucleic acid 121 are evolutionarily related. In some embodiments, the sequences in the first nucleic acid 111 and the second nucleic acid 121 between the two conserved regions 101 and 103 are different such that the difference can be usedto distinguish the first organism 110 and the second organism 120. In some embodiments, the first nucleic acid 111 and the second nucleic acid 121 are considered one identifying nucleic acid shared by the first organism 110 and the second organism 120. In some embodiments, nucleic acids 111 and 113 have the same sequences in the conserved regions 101 / 103, and can both be amplified by a PCR reaction with a pair of primers having no degeneracy. In some embodiments, nucleic acids 111 and 121 have highly similar sequences in the conserved region 101 / 103, and can both be amplified by a PCR reaction with a pair of primers having some degrees of degeneracy. Since the primer pairs binding to the conserved regions 101 and 103 can produce PCR signal for both the first organism and the second organism, the first organism and the second organism are considered to be in the same group ("the first group"). In some embodiments, the first group is a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.

[0096] Similarly, in some embodiments, the third organism 210 include a third nucleic acid 211, and the fourth organism include a fourth nucleic acid 221. The third organism and the fourth organism are of different species or strains and have different sequence between the third nucleic acid 211 and the fourth nucleic acid 221. The third nucleic acid 211 and the fourth nucleic acid 221, however, both include the conserved regions 201 and 203. In some embodiments, the third nucleic acid 211 and the fourth nucleic acid 221 are evolutionarily related. In some embodiments, the sequences in the third nucleic acid 211 and the fourth nucleic acid 221 between the two conserved regions 201 and 203 are different such that the difference can be used to distinguish the third organism 210 and the fourth organism 220. In some embodiments, the first nucleic acid 111 and the second nucleic acid 121 are considered one identifying nucleic acid shared by the first organism 110 and the second organism 120. In some embodiments, nucleic acids 211 and 221 have the same sequences in the conserved region 201 / 203, and can both be amplified by a PCR reaction with a pair of primers having no degeneracy. In some embodiments, nucleic acids 211 and 213 have highly similar sequences in the conserved region 201 / 203, and can both be amplified by a PCR reaction with a pair of primers having some degrees of degeneracy. Since the primer pairs binding to the conserved regions 201 and 203 can produce PCR signal for both the third organism and the fourth organism, the third organism and the fourth organism are considered to be in the same group("the second group"). In some embodiments, the second group is a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.

[0097] In some embodiments, since the members of the first group and the second group are from different taxonomic domain, there is no overlapping between the first group and the second group.

[0098] In some embodiments, each pair of the primers used in the first round of PCR (see e.g., primers 151, 153, 251 and 253 of Fig. IB) comprises a pair of overhang regions specific to one of the category of microorganisms, such that a unique barcode can be attached to amplicons derived from that particular group of microorganisms.

[0099] In some embodiments, the method further comprises attaching a barcoding sequence to the amplicon. In some embodiments, attaching the barcoding sequence comprises attaching the barcoding sequence with another round of polymerase chain reaction. In some embodiments, the barcoding sequence is unique for a group of microorganisms. For example, in some embodiments, the barcoding sequence distinguishes different species, different genus, different families, different orders, different orders, different phylum, different kingdoms, different domains, or two or more arbitrary groups. In some embodiments, the barcoding sequence distinguishes the different groups on the basis of the different sequences of the primer pairs used in the first round of PCR.

[0100] The selection of the nucleic acids with the two conserved regions are not limited, as long as the two conserved regions exist in all or a significant number of species / strains of the particular group. Non-limiting examples of suitable nucleic acids include 16S rRNA genes of species / strains in the bacteria domain, or 18s rRNA genes, ITS1 rRNA sequences, 5.8S genes, ITS2 RNA sequence, 28S rRNA genes. These rRNA genes are sometimes chosen as they often include several conserved regions, which makes design of primer pairs convenient.

[0101] In some embodiments, the nucleic acids are DNA molecules, such as genomic DNA molecules. In some embodiments, the nucleic acids are RNA molecules.

[0102] In some embodiments, the two conserved regions are about 1,250 base pairs apart or more, such as about 1,500 base pairs apart or more, about 2,000 base pairs or more, about 2,500 base pairs or more, about 3,000 base pairs or more, about 4,000 base pairs or more, or about 5,000 base pairs or more. The further the two conserved regions from each other, the longer thenucleic acid could be sequenced, which would makes distinguishing closely related species / strains easier.Removal of Non-Microbial Nucleic Acid

[0103] In some embodiments, the method herein further comprises removing a nucleic acid that does not belong to the microorganism assemblage, but are potentially amplifiable by the multiplex PCR reaction.

[0104] For example, in the case of a sample from a metazoan host or vector, or a sample comprising a plant material (such as an agricultural sample), since the host / vector / plant nucleic acids amplifiable by the multiplex PCR can exist in substantial quantity or even of far greater quantity than the microbial nucleic acids amplifiable by the multiplex PCR, the host / vector / plant nucleic acids could compete for primer and polymerase in the PCR reaction and cause insufficient amplification of the microbial nucleic acids intended to be amplified. This is especially problematic when eukaryotic ribosomal region (18S, 5S, ITS, 28S), which sometimes have similar sequences in both the host / vector / plant and the microorganisms of interest, is being amplified. Completely or partially removing the non-microbial nucleic acid can eliminate or at least reduce the above issues.

[0105] In some embodiments, the non-microbial nucleic acid is removed by a gene editing method. In some embodiments, the genome editing method includes a CRISPR-based method, such as a CRISPR / Cas9 method.

[0106] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a “seed” sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.

[0107] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The Reel domain binds the guide RNA, while the Bridge helix binds to targetDNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.

[0108] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Patent Appl. Publ. No. US2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0109] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.

[0110] In some embodiments, the CRISPR / Cas gene editing method is designed to target one or more of the following sequences (such as by designing the gRNA to target these sequences):Multiplex Polymerase Chain Reaction (PCR)

[0111] In some embodiments, the selected nucleic acids having identifiable characteristics in the organisms of interest in the sample are amplified using primers designed for predetermined organism groups by PCR reaction, the product of which are referred to as "amplicons." The amplified nucleic acids are suitable for being sequenced and analyzed in the subsequent steps.

[0112] In some embodiments, each primer in a primer pair includes a target binding sequence for binding to the conserved regions of the nucleic acids in one group of organisms. In some embodiments, each primer pair further includes a pair of barcoding sequences for identification purposes.

[0113] Some exemplary non-limiting target binding sequences for used in the primer pairs are provided in Table 1 below:

[0114] Fig. IB illustrates some aspects of the multiplex polymerase chain reaction, in accordance with some embodiments.

[0115] Referring to Fig. IB, in some embodiments, the sample 10 including the nucleic acids 111, 121, 211 and 221 are amplified by a multiplexing PCR with a first primer pair including primers 151 and 153 for amplifying nucleic acids including conserved regions 101 and 103; and a second primer pair including primers 251 and 253 for amplifying nucleic acids including conserved regions 201 and 203.

[0116] Referring to Fig. IB, in some embodiments, primers 151 and 153 include a first barcode pair (the stars attached to primers 151 and 153), and primers 251 and 253 include a second barcode pair (the heptagons attached to primers 251 and 253).

[0117] Referring to Fig. IB, in some embodiments, the product 20 of the multiplexing PCR includes amplicons of sample 10, which includes amplified first nucleic acid 111’ including the first barcode pair, amplified second nucleic acid 121’ including the first barcode pair, amplified third nucleic acid 21 1 ’ including the second barcode pair, and amplified fourth nucleic acid 221 ’ including the second barcode pair. It is worth noting that, although in Fig. IB the product 20 is shown to include only four types of nucleic acids which are grouped according to the types, product 20 in fact can include hundreds or thousands of different types of nucleic acids, and the different types of nucleic acids are homogeneously intermixed.Sequencing

[0118] In some embodiments, a sequencing is performed on the PCR product to produce raw sequencing data including sequencing reads of the amplicons in the PCR product.

[0119] Since the product includes mixture of hundreds or thousands of types of amplicons, in some embodiments the sequencing is a high throughput sequencing. Furthermore, since organisms in the same group are related and may have similar nucleic acid sequences between the conserved regions used for amplification, it is desirable that the sequencing be a high-fidelity sequencing, which produces highly accurate results. Examples of high-fidelity sequencing methods include circular sequencing, such as the circular consensus sequencing.

[0120] Circular sequencing is described in, for example, Wenger et al. (Nature Biotechnology volume 37, pagesl 155-1162 (2019)), and are commercially available from, for example, Pacific Biosciences. Briefly, circular sequencing converts a linear double stranded DNA molecule to be sequenced into a single stranded circular DNA molecule by ligating 5’-end of the first stand to 3 ’-end of the second strand, and 3 ’-end of the first stand to 5 ’-end of the second strand with adapters. The single stranded circular DNA molecule is then used as a template for the polymerase to make a long single stranded DNA molecule including several passes of the template sequence. Since sequencing the long single stranded DNA molecule reads through the several copies of the template sequence included in the long single stranded DNA molecule, the sequencing results include several passes of the original sequence. This redundancy of sequence information allows the correction of sequencing errors caused by the polymerase or during the sequence reading which, if occurs in one of the passes, likely do not repeat in other passes. In some embodiments, the sequencing of the long single stranded DNA molecule reads 20,000 bp or more, such as 30,000 bp or more, 40,000 bp or more, 50,000 bp or more, 60,000 bp or more, 70,000 bp or more, 80,000 bp or more, 90,000 bp or more, or 100,000 bp or more.Analysis of Raw Sequencing Data and Identification of Organisms

[0121] In some embodiments, the sequencing results are analyzed, using for example bioinformatics, so that the organisms in the sample can be identified.

[0122] In some embodiments, analyzing the sequencing results includes analyzing the sequencing results with an edge graphing algorithm, such as based on the circular consensus sequencing (CCS) reads.

[0123] In some embodiments, the method includes comparing the sequencing reads (or processed sequencing results thereof) obtained in the sequencing step with a nucleic acid sequence database (such as a DNA sequence database). In some embodiments, the method includes performing sequence alignment on the sequencing reads (or processed sequencing results thereof) against known nucleic acid sequences of organisms, such as known nucleic acid sequences from a nucleic acid sequence database.

[0124] The process of analyzing raw sequencing data obtained from a multiplex PCR is described in, for example, Earl et al. (Microbiome . 6 : 190, 2018). The entirety of the reference is hereby incorporated herein by reference.

[0125] Referring to Fig. 1C, in some embodiments, analyzing the sequence includes a preclustering step 310 and a clustering step 350.

[0126] In some embodiments, the pre-clustering step 310 includes generating demultiplexed sequencing reads.

[0127] In some embodiments, generating demultiplexed sequencing reads includes discarding sequencing reads having insufficient passes 311. In some embodiments, the passing number is considered insufficient if it is less than 4 passes of the sequenced template sequence, such as less than 5 passes, less than 6 passes, or less than 7 passes.

[0128] In some embodiments, generating demultiplexed sequencing reads further includes demultiplexing the sequencing reads 313 according to the barcode sequence incorporated into the sequence from the primer during the multiplex PCR step.

[0129] In some embodiments, the pre-clustering step includes labeling and combining sequencing reads 315.

[0130] In some embodiments, the pre-clustering step includes assigning taxonomy to each sequencing read 317.

[0131] In some embodiments, the pre-clustering step includes filtering the sequencing reads 319.

[0132] In some embodiments, filtering the sequencing reads 319 includes removing sequencing reads having a size outside a predetermined value. One of ordinary skill in the art would be able to estimate the length of sequence reads based on the primer pairs used and the selection of nucleic acids for amplification. Therefore, in some embodiments, removing sequencing reads having a size outside a predetermined value includes removing sequencing reads significantly longer than the estimated value, such as 20% or more, 50% or more, 100% or more, 150% or more, or 200% or more. In some embodiments, removing sequencing reads having a size outside a predetermined value includes removing sequencing reads significantly shorter than the estimated value, such as 80% or less, 60% or less, 50% or less, or 40% or less of the estimated value.

[0133] In some embodiments, filtering the sequencing reads 319 includes removing reads that include host sequences. As described elsewhere herein, in certain embodiments the method identifies an organism in a microbiome colonizing a particular environment within the body or on the surface of the body of a subject. According to these embodiments, any nucleic acidsequence derived from the subject most likely does not belong to any organisms in the microbiome and should be removed.

[0134] In some embodiments, filtering the sequencing reads 319 includes removing sequencing reads that do not include the sequence of one or both primers of the primer pair.

[0135] In some embodiments, filtering the sequencing reads 319 includes sequencing reads that includes the sequences of the primers, but the orientation of one or both primers is incorrect.

[0136] In some embodiments, the sequencing reads obtained in pre-clustering are filtered based on cumulative expected error, and used for the clustering step 350.

[0137] In some embodiments, the clustering step 350 clusters the sequencing reads into operational taxonomic units (OTU). As used herein, the term "operational taxonomic unit" refers to a cluster of organisms grouped by DNA sequence similarity of a specific percentage nucleotide sequence. In some embodiments, two organisms are considered to be in the same OTU if the sequence of the identifiable nucleic acid of the organisms have 97% similarity or more, such as 98% similarity or more, 99% similarity or more, or 100% similarity (identical).

[0138] In some embodiments, the clustering step 350 includes collapsing and sorting identical by abundance 351, which identifies unique sequences.

[0139] In some embodiments, the clustering step 350 includes clustering sequencing reads into OTUs 353.

[0140] In some embodiments, the clustering step 350 includes filtering out chimera reads 355.

[0141] In some embodiments, the clustering step 350 includes creating an OTU count table 358 by mapping the filtered results from the end of the pre-clustering step (filtering reads 319).

[0142] In some embodiments, the clustering step 350 includes classifying each OTU taxonomically based on a representative "centroid" sequence 357.

[0143] In some embodiments, the clustering step 350 includes determining amplicon sequence variants (ASVs) and / or identifying the species or strains organisms in the sample 359.Kit for Identifying Organisms Across Multiple Taxonomic Domains in Sample

[0144] In some embodiments, the present disclosure is directed to a kit for identifying organisms across multiple taxonomic domains in a sample.

[0145] In some embodiments, the kit is for performing the methods herein, such as the methods described in the "Method of Identifying Organisms Across Multiple Taxonomic Domains in Sample" section.

[0146] In some embodiments, the kit includes a plurality of primer pairs.

[0147] In some embodiments, the primer pairs in the plurality of primer pairs are the same as or similar to those described herein, such as in the "Method of Identifying Organisms Across Multiple Taxonomic Domains in Sample" section.

[0148] In some embodiments, the plurality of primer pairs includes a primer pair for amplifying nucleic acids having identifiable characteristics in the bacteria domain, and a primer pair for amplifying nucleic acids having identifiable characteristics in the eukarya domain.

[0149] In some embodiments, the plurality of primer pairs includes at least two selected from the group consisting of: a primer pair for amplifying nucleic acids having identifiable characteristics in bacteria, a primer pair for amplifying nucleic acids having identifiable characteristics in fungi, and a primer pair for amplifying nucleic acids having identifiable characteristics in unicellular eukaryotic parasites.Examples

[0150] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1

[0151] In Example 1, experimental protocols used for identifying microorganisms across several taxonomic domains based on the bacterial 16S ribosomal RNA gene sequences and one or more of eukaryotic genes according to some embodiments herein is described.Reagents and Sources

[0152] The reagents used in the experiments, as well as the sources thereof, are listed below:First PCR Amplification of Full-length 16S rRNA Genes (VI -V9 Regions)

[0153] A first round PCR, which serves as the initial amplification of full-length 16s ribosomal RNA genes of microorganisms in the sample, was performed.

[0154] The PCR master mix was prepared according to the table below:* The “x 1.25” means the master mix was prepared with 25% overage

[0155] 15 pL aliquots of the PCR master mix were then transfer across a 96-well plate. After estimating the DNA with nanodrop, 1-10 pL DNA sample (500 ng) was added to the individual well and the volume was adjusted to 25 pL with water.

[0156] The plates were sealed thoroughly with an adhesive seal to prevent evaporation during PCR, and were vortexed and briefly spined in a refrigerated centrifuge (4°C) to ensure that the entire sample volume is at the bottom of each well.

[0157] The PCR reaction was then performed according to the following cycling parameters:

[0158] The amplicons were tested by agarose gel electrophoresis. 5 pL product was mixed with 1 pL of 6x loading buffer from each well and loaded into a 1% agarose gel. To determine the size of the products, Ikb DNA ladder was run in the same gel along with the amplicons.

[0159] The gel images were analyzed with the ImageJ program to normalize the concentrations of the amplicons for the nested PCR with the barcoded primers.

[0160] The expected target amplicon size is ~1500 bp, and the amount of amplicon material generated from each sample should be comparable as assessed by analyzing the relative intensity of the -1500 bp PCR product on an agarose electrophoresis gel.Second PCR Amplification

[0161] A second round PCR was performed to further amplify the bacterial 16s rRNA gene and the fungal and / or parasite gene amplified in the first round PCR, as well as to attach barcode sequences to the amplicons.

[0162] The master mix for the second round PCR was prepared according to the following table:

[0163] 1-10 pL of the amplicon was added to the individual well (the exact volumes were determined based on the estimation of amplicon amount with the imageJ) and the volume was adjusted to 22.5 pL with water.

[0164] The plates were sealed thoroughly with an adhesive seal to prevent evaporation during PCR, and vortexed and briefly spined in a refrigerated centrifuge (4°C) to ensure that the entire sample volume is at the bottom of each well.

[0165] The second PCR was performed using the cycling parameters set forth in the Table below:

[0166] The second PCR resulted in barcoded amplicons.AMPure PB Bead Purification of Pooled Barcoded Amplicons

[0167] The volume of the pooled samples was determined, and 0.60X of AMPure PB beads were added to the sample in a test tube. The bead / DNA mixtures were mixed thoroughly by finger tapping or pipetting. The tube was placed on an end-over-end rotator for 10 minutes at room temperature to allow the DNA in the tube to bind to beads.

[0168] The tube was spined down briefly (e.g., for about 1 second) to collect the beads. The tube was placed in a magnetic rack until the beads collect to the side of the tube and the solution appeared clear. With the tube still on the magnetic rack, the cleared supernatant was slowly pipette off and transferred to another tube.

[0169] The beads were washed with freshly prepared 80% ethanol. Specifically, 180 pl of 80% ethanol was slowly dispensed against the side of the tube opposite the beads. The tube was allowed to sit for about 30 seconds without allowing the bead pellet to be disturbed. After 30 seconds, the 80% ethanol was pipetted and discarded. The beads were then washed for a second time following the same procedures.

[0170] The tube was removed from the magnetic rack and spin it to pellet the beads. The tube was then placed back on the magnetic rack and any remaining 80% ethanol was removed. The tube was removed from the magnetic rack and the beads were allowed to air-dry (with the tube cap open) for 60 seconds.

[0171] 30 pL of elution buffer was added to the tube, which was incubated at room temperature for about 2 minutes to allow the DNA to be eluted from the beads. The tube was briefly spined and placed back on the magnetic rack, and the beads were allowed to separate fully. The supernatant was transfer to a new DNA Lo-Bind tube without disturbing the beads.Measure DNA Concentration using Qubit Fluorometer.

[0172] About 1 pL of the eluted DNA was aliquoted and diluted by 1 : 10 with IX elution buffer. The DNA concentration in the diluted sample was measured using Qubit dsDNA High Sensitivity Kit reagents.

[0173] Based on the measured concentrations, all the purified samples were pulled together into equimolar concentration to make a SMRTbell library.Enumerated Embodiments

[0174] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0175] Embodiment 1 : A method of identifying microorganisms across multiple taxonomic domains in a sample, the method comprising: amplifying one or more identifying nucleic acids of a plurality of microorganisms across multiple taxonomic domains in the sample with a multiplex polymerase chain reaction (PCR) using a plurality of primer pairs, thereby generating amplicons of a plurality of microorganisms present in the sample; performing sequencing on the amplicons to obtain sequencing reads of the amplicons; and identifying the plurality of microorganisms in the sample based on the sequencing reads, wherein: a first identifying nucleic acid present in a first group of microorganisms from a first taxonomic domain is amplified by a first primer pair of the plurality of primer pairs, and the first primer pair binds to two conserved regions at two ends of the at least one first identifying nucleic acid shared by the microorganisms in the first group, and a second identifying nucleic acid present in a second group of microorganisms from a second taxonomic domain is amplified by a second primer pair of the plurality of primer pairs, and the second primer pair binds to two conserved regions at two ends of the at least one second identifying nucleic acid shared by the microorganisms in the second group, wherein the sample is at least one selected from a metazoan sample (optionally an animal host sample or a vector sample), a plant sample (optionally a plant host sample or an agricultural sample), and an environmental sample (such as a sample from an industrial site or the nature), and wherein the multiple taxonomic domains comprise a prokaryotic domain and Eukaryotes.

[0176] Embodiment 2: The method of Embodiment 1, further comprising attaching a first barcode sequence to the amplicon from the microorganisms from the first group, and attaching a second barcode sequence to the amplicon from the microorganisms from the second group.

[0177] Embodiment 3: The method of any one of Embodiments 1-2, wherein at least one of the following applies:(a) the plurality of primer pairs does not amplify any identifying nucleic acid in the host;(b) the method further comprises removing a nucleic acid from a non-microbial source amplifiable by two or more primers of the plurality of primer pairs from the sample.

[0178] Embodiment 4: The method of Embodiment 3, wherein the nucleic acid from a non- microbial source is a nucleic acid from a metazoan or plant species serving as the host or the vector of the microorganisms.

[0179] Embodiment 5: The method of Embodiment 4, wherein the nucleic acid from a non- microbial is removed by a gene editing method.

[0180] Embodiment 6: The method of any one of Embodiments 1-5, wherein the group of microorganisms comprises organisms in a taxonomic domain, a kingdom, a phylum, a class, an order, or a family.

[0181] Embodiment 7: The method of any one of Embodiments 1-6, wherein the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the bacterial domain and at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the eukaryal domain.

[0182] Embodiment 8: The method of any one of Embodiments 1-7, wherein the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of bacteria, at least one primer pair amplifying at least one identifying nucleic acid of fungi, or at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

[0183] Embodiment 9: The method of any one of Embodiments 1-8, wherein the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Amoebozoa parasite, and an Alveolate parasite.

[0184] Embodiment 10: The method of any one of Embodiments 8-9, whereinthe plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acids of bacteria, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 1 and a second primer comprising the sequence set forth in SEQ ID NO: 1, a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4, a first primer comprising the sequence set forth in SEQ ID NO:5 and a second primer comprising the sequence set forth in SEQ ID NO:6, or a first primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO:8, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of fungi, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO:9 and a second primer comprising the sequence set forth in SEQ ID NO: 10, or the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO: 12.

[0185] Embodiment 11 : The method of any one of Embodiments 1-10 wherein the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

[0186] Embodiment 12: The method of Embodiment 11, wherein the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene of species / strains in the eukarya domain, ITS! rRNA sequences, 5.8S genes, ITS2 RNA sequence and 28S rRNA genes, or any combinations thereof.

[0187] Embodiment 13: The method of any one of Embodiments 1-12, wherein the plurality of primer pairs is configured that the amplicons of the plurality of microorganisms generated by the multiplex PCR has a minimal length of 1,300 base pairs.

[0188] Embodiment 14: The method of any one of Embodiments 1-13, wherein the host is a mammal, optionally a human.

[0189] Embodiment 15: The method of Embodiment 14, wherein the sample obtained from the host comprises at least one selected from the group consisting of a blood sample, a bronchial alveolar lavage specimen, a bile duct specimen, a cerebrospinal fluid (CSF) sample, a dental sample, a fecal sample, a gastrointestinal aspirate specimen, a medical implant specimen, an indwelling pump specimen, a middle-ear effusion specimen, a mucosal sample, an ocular specimen, a plasma specimen, periodontal crevicular specimen, a saliva sample, a serum specimen, a skin swab specimen, a surgical debridement specimen, a synovial fluid sample, a tissue biopsy sample, a urine sample, and a wound specimen.

[0190] Embodiment 16: The method of Embodiment 15, wherein the dental sample comprises at least one selected from the group consisting of a caries specimen, a dental implant specimen, a dental plaque specimen, an endodontic pulp specimen, a periodontal crevicular specimen, a periodontal tissue biopsy specimen, and a salivary specimen.

[0191] Embodiment 17: The method of any one of Embodiments 1-13, wherein the vector is an arthropod vector of infectious disease, optionally an arachnid sample or an insect sample.

[0192] Embodiment 18: The method of Embodiment 17, wherein the arachnid sample comprises or is derived from at least one selected from the group consisting of a tick, a spider, a mite, and a chigger, or wherein the insect sample comprises or is derived from at least one selected from the group consisting of a mosquito, a biting fly, a flea, a louse, a bed bug, a bat bug, and a sand fly.

[0193] Embodiment 19: The method of any one of Embodiments 1-13, wherein the sample is an environment sample, a veterinary sample, an agricultural sample, an aquaculture sample, an industrial sample, or combinations thereof.

[0194] Embodiment 20: The method of any one of Embodiments 1-19, wherein performing sequencing on the amplicons of the sample comprises performing circular consensus sequencing on the amplicons.

[0195] Embodiment 21 : The method of any one of Embodiments 1-20, wherein identifying microorganisms in the sample based on results of the sequencing comprises comparing sequencing reads obtained in the sequencing step or processed sequencing results thereof with a nucleic acid sequence database.

[0196] Embodiment 22: The method of any one of Embodiments 1-21, wherein identifying microorganisms in the sample based on results of the sequencing comprises performing sequencealignment on the sequencing reads or processed sequencing results thereof against known nucleic acid sequences of microorganisms.

[0197] Embodiment 23 : A kit, comprising: a first primer pair for amplifying at least one identifying nucleic acid in a first group of microorganisms; and a second primer pair for amplifying at least one identifying nucleic acid in a second group or microorganisms, wherein: the first group of microorganisms and the second group of microorganisms are from different taxonomic domains, the first primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the first group of microorganisms, the second primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the second group of microorganisms, each primer in the first primer pair and the second primer pair comprises a barcoding sequence unique to the primer pair the primer belongs to, and the first primer pair and the second primer pair are configured such that no identifying nucleic acid from a host of the first group of microorganisms or the second group of microorganisms are amplified by the primer pairs.

[0198] Embodiment 24: The kit of Embodiment 23, further comprises a first barcode for attaching to amplicons generated by the first primer pair and a second barcode for attaching to amplicons generated by the second primer pair.

[0199] Embodiment 25: The kit of any one of Embodiments 23-24, wherein the first group or the second group comprises microorganisms in a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.

[0200] Embodiment 26: The kit of any one of Embodiments 23-25, wherein the first primer pair amplifies at least one identifying nucleic acid of microorganisms in the bacteria domain and the second primer pair amplifies at least one identifying nucleic acid of microorganisms in the eukarya domain.

[0201] Embodiment 27: The kit of any one of Embodiments 23-26, wherein the first primer pair and the second primer pair comprises at least two selected from the group consisting of aprimer pair amplifying at least one identifying nucleic acid of bacteria, a primer pair amplifying at least one identifying nucleic acid of fungi, and a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

[0202] Embodiment 28: The kit of any one of Embodiments 23-27, wherein at least one of the first primer pair or the second primer pair amplifies at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Aloebozoa parasite, and an Alveolate parasite.

[0203] Embodiment 29: The kit of any one of Embodiments 23-28, wherein the first primer pair and the second primer comprises at least two selected from the group consisting of:(a) a primer pair amplifying at least one identifying nucleic acid of bacteria, comprising: a first primer comprising the sequence set forth in SEQ ID NO: 1 and a second primer comprising the sequence set forth in SEQ ID NO: 1, a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4, a first primer comprising the sequence set forth in SEQ ID NO:5 and a second primer comprising the sequence set forth in SEQ ID NO:6, or a first primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO: 8;(b) a primer pair amplifying at least one identifying nucleic acid of fungi, comprising a first primer comprising the sequence set forth in SEQ ID NO:9 and a second primer comprising the sequence set forth in SEQ ID NO: 10; and(c) a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, comprising a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO: 12.

[0204] Embodiment 30: The kit of any one of Embodiments 23-29, wherein the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

[0205] Embodiment 31 : The kit of Embodiment 30, wherein the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene, a ITS1 rRNA sequence,a 5.8S gene, an TTS2 RNA sequence, a 28S rRNA gene of species / strains in the eukarya domain, or combinations thereof.

[0206] Embodiment 32: The kit of any one of Embodiments 23-31 wherein an amplicon amplified by the first primer pair or the second primer pair has a length of 1,250 base pair or longer.

[0207] Embodiment 33: The kit of any one of Embodiments 23-33, further comprising a polynucleic acid adaptor for converting an amplicon of a PCR to a circular DNA for circular consensus sequencing.

[0208] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying microorganisms across multiple taxonomic domains in a sample, the method comprising: amplifying one or more identifying nucleic acids of a plurality of microorganisms across multiple taxonomic domains in the sample with a multiplex polymerase chain reaction (PCR) using a plurality of primer pairs, thereby generating amplicons of a plurality of microorganisms present in the sample; performing sequencing on the amplicons to obtain sequencing reads of the amplicons; and identifying the plurality of microorganisms in the sample based on the sequencing reads, wherein: a first identifying nucleic acid present in a first group of microorganisms from a first taxonomic domain is amplified by a first primer pair of the plurality of primer pairs, and the first primer pair binds to two conserved regions at two ends of the at least one first identifying nucleic acid shared by the microorganisms in the first group, and a second identifying nucleic acid present in a second group of microorganisms from a second taxonomic domain is amplified by a second primer pair of the plurality of primer pairs, and the second primer pair binds to two conserved regions at two ends of the at least one second identifying nucleic acid shared by the microorganisms in the second group, wherein the sample is at least one selected from a metazoan sample (optionally an animal host sample or a vector sample), a plant sample (optionally a plant host sample or an agricultural sample), and an environmental sample (such as a sample from an industrial site or the nature), and wherein the multiple taxonomic domains comprise a prokaryotic domain and Eukaryotes.

2. The method of claim 1, further comprising attaching a first barcode sequence to the amplicon from the microorganisms from the first group, and attaching a second barcode sequence to the amplicon from the microorganisms from the second group.

3. The method of any one of claims 1-2, wherein at least one of the following applies:(a) the plurality of primer pairs does not amplify any identifying nucleic acid in the host;(b) the method further comprises removing a nucleic acid from a non-microbial source amplifiable by two or more primers of the plurality of primer pairs from the sample.

4. The method of claim 3, wherein the nucleic acid from a non-microbial source is a nucleic acid from a metazoan or plant species serving as the host or the vector of the microorganisms.

5. The method of claim 4, wherein the nucleic acid from a non-microbial is removed by a gene editing method.

6. The method of any one of claims 1-5, wherein the group of microorganisms comprises organisms in a taxonomic domain, a kingdom, a phylum, a class, an order, or a family.

7. The method of any one of claims 1-6, wherein the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the bacterial domain and at least one primer pair amplifying at least one identifying nucleic acid of microorganisms in the eukaryal domain.

8. The method of any one of claims 1-7, wherein the plurality of primer pairs comprises at least one primer pair amplifying at least one identifying nucleic acid of bacteria, at least one primer pair amplifying at least one identifying nucleic acid of fungi, or at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

9. The method of any one of claims 1-8, wherein the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Amoebozoa parasite, and an Alveolate parasite.

10. The method of any one of claims 8-9, whereinthe plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acids of bacteria, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 1 and a second primer comprising the sequence set forth in SEQ ID NO: 1, a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4, a first primer comprising the sequence set forth in SEQ ID NO:5 and a second primer comprising the sequence set forth in SEQ ID NO:6, or a first primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO:8, the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of fungi, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO:9 and a second primer comprising the sequence set forth in SEQ ID NO: 10, or the plurality of primer pairs comprises the at least one primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, and the at least one primer pair comprises: a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO: 12.

11. The method of any one of claims 1-10 wherein the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

12. The method of claim 11, wherein the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene of species / strains in the eukarya domain, ITS1 rRNA sequences, 5.8S genes, ITS2 RNA sequence and 28S rRNA genes, or any combinations thereof.

13. The method of any one of claims 1-12, wherein the plurality of primer pairs is configured that the amplicons of the plurality of microorganisms generated by the multiplex PCR has a minimal length of 1,300 base pairs.

14. The method of any one of claims 1-13, wherein the host is a mammal, optionally a human.

15. The method of claim 14, wherein the sample obtained from the host comprises at least one selected from the group consisting of a blood sample, a bronchial alveolar lavage specimen, a bile duct specimen, a cerebrospinal fluid (CSF) sample, a dental sample, a fecal sample, a gastrointestinal aspirate specimen, a medical implant specimen, an indwelling pump specimen, a middle-ear effusion specimen, a mucosal sample, an ocular specimen, a plasma specimen, periodontal crevicular specimen, a saliva sample, a serum specimen, a skin swab specimen, a surgical debridement specimen, a synovial fluid sample, a tissue biopsy sample, a urine sample, and a wound specimen.

16. The method of claim 15, wherein the dental sample comprises at least one selected from the group consisting of a caries specimen, a dental implant specimen, a dental plaque specimen, an endodontic pulp specimen, a periodontal crevicular specimen, a periodontal tissue biopsy specimen, and a salivary specimen.

17. The method of any one of claims 1-13, wherein the vector is an arthropod vector of infectious disease, optionally an arachnid sample or an insect sample.

18. The method of claim 17, wherein the arachnid sample comprises or is derived from at least one selected from the group consisting of a tick, a spider, a mite, and a chigger, or wherein the insect sample comprises or is derived from at least one selected from the group consisting of a mosquito, a biting fly, a flea, a louse, a bed bug, a bat bug, and a sand fly.

19. The method of any one of claims 1-13, wherein the sample is an environment sample, a veterinary sample, an agricultural sample, an aquaculture sample, an industrial sample, or combinations thereof.

20. The method of any one of claims 1-19, wherein performing sequencing on the amplicons of the sample comprises performing circular consensus sequencing on the amplicons.

21. The method of any one of claims 1-20, wherein identifying microorganisms in the sample based on results of the sequencing comprises comparing sequencing reads obtained in the sequencing step or processed sequencing results thereof with a nucleic acid sequence database.

22. The method of any one of claims 1-21, wherein identifying microorganisms in the sample based on results of the sequencing comprises performing sequence alignment on the sequencing reads or processed sequencing results thereof against known nucleic acid sequences of microorganisms.

23. A kit, comprising: a first primer pair for amplifying at least one identifying nucleic acid in a first group of microorganisms; and a second primer pair for amplifying at least one identifying nucleic acid in a second group or microorganisms, wherein: the first group of microorganisms and the second group of microorganisms are from different taxonomic domains, the first primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the first group of microorganisms, the second primer pair binds to two conserved regions shared by the at least one identifying nucleic acid in the second group of microorganisms, each primer in the first primer pair and the second primer pair comprises a barcoding sequence unique to the primer pair the primer belongs to, and the first primer pair and the second primer pair are configured such that no identifying nucleic acid from a host of the first group of microorganisms or the second group of microorganisms are amplified by the primer pairs.

24. The kit of claim 23, further comprises a first barcode for attaching to amplicons generated by the first primer pair and a second barcode for attaching to amplicons generated by the second primer pair.

25. The kit of any one of claims 23-24, wherein the first group or the second group comprises microorganisms in a taxonomic domain, a kingdom, a phylum, a class, an order, a family, or portions thereof.

26. The kit of any one of claims 23-25, wherein the first primer pair amplifies at least one identifying nucleic acid of microorganisms in the bacteria domain and the second primer pair amplifies at least one identifying nucleic acid of microorganisms in the eukarya domain.

27. The kit of any one of claims 23-26, wherein the first primer pair and the second primer pair comprises at least two selected from the group consisting of a primer pair amplifying at least one identifying nucleic acid of bacteria, a primer pair amplifying at least one identifying nucleic acid of fungi, and a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites.

28. The kit of any one of claims 23-27, wherein at least one of the first primer pair or the second primer pair amplifies at least one identifying nucleic acid of unicellular eukaryotic parasites, and wherein the unicellular eukaryotic parasites comprise at least one selected from the group of an Apicoplexans parasite, a Kinetoplastids parasite, an Aloebozoa parasite, and an Alveolate parasite.

29. The kit of any one of claims 23-28, wherein the first primer pair and the second primer comprises at least two selected from the group consisting of(a) a primer pair amplifying at least one identifying nucleic acid of bacteria, comprising: a first primer comprising the sequence set forth in SEQ ID NO: 1 and a second primer comprising the sequence set forth in SEQ ID NO: 1, a first primer comprising the sequence set forth in SEQ ID NO:3 and a second primer comprising the sequence set forth in SEQ ID NO:4,a first primer comprising the sequence set forth in SEQ ID NO:5 and a second primer comprising the sequence set forth in SEQ ID NO: 6, or a first primer comprising the sequence set forth in SEQ ID NO:7 and a second primer comprising the sequence set forth in SEQ ID NO:8;(b) a primer pair amplifying at least one identifying nucleic acid of fungi, comprising a first primer comprising the sequence set forth in SEQ ID NON and a second primer comprising the sequence set forth in SEQ ID NO: 10; and(c) a primer pair amplifying at least one identifying nucleic acid of unicellular eukaryotic parasites, comprising a first primer comprising the sequence set forth in SEQ ID NO: 11 and a second primer comprising the sequence set forth in SEQ ID NO: 12.

30. The kit of any one of claims 23-29, wherein the at least one identifying nucleic acid of at least one group of microorganisms comprises a ribosomal RNA (rRNA) gene.

31. The kit of claim 30, wherein the rRNA gene comprises a 16S rRNA gene of species / strains in the bacteria domain, a 18s rRNA gene, a ITS1 rRNA sequence, a 5.8S gene, an ITS2 RNA sequence, a 28S rRNA gene of species / strains in the eukarya domain, or combinations thereof.

32. The kit of any one of claims 23-31 wherein an amplicon amplified by the first primer pair or the second primer pair has a length of 1,250 base pair or longer.

33. The kit of any one of claims 23-33, further comprising a polynucleic acid adaptor for converting an amplicon of a PCR to a circular DNA for circular consensus sequencing.

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