WO2014197607A1 - Détection de maladies transmises par les tiques - Google Patents

Détection de maladies transmises par les tiques Download PDF

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Publication number
WO2014197607A1
WO2014197607A1 PCT/US2014/040922 US2014040922W WO2014197607A1 WO 2014197607 A1 WO2014197607 A1 WO 2014197607A1 US 2014040922 W US2014040922 W US 2014040922W WO 2014197607 A1 WO2014197607 A1 WO 2014197607A1
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tick
nucleic acid
microchip
borne disease
borrelia
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PCT/US2014/040922
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English (en)
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Charles Chiu
Andrea SWEI
Deanna LEE
Samia NACCACHE
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The Regents Of The University Of California
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Publication of WO2014197607A1 publication Critical patent/WO2014197607A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/29—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Richettsiales (o)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/45—Toxoplasma

Definitions

  • Lyme disease is a serious chronic borrelial infection that can be characterized by a diversity of
  • symptoms at various stages Approximately 3 to 14 days following the initiating tick bite, symptoms may include fever, flu-like illness, and the appearance of the Erythema Migrans (EM) skin rash. Stage two, occurring weeks to months after the initial bite, can include further skin involvement, arthritis, nervous system complaints, and cardiac pathology. Stage three can be characterized by more severe arthritis and nervous system complications.
  • EM Erythema Migrans
  • the disclosure provides for a method of detecting a tick-borne disease
  • the method comprising: immobilizing one or more polynucleotide molecules at a plurality of positions on a substrate, wherein the one or more polynucleotide molecules relate to genomic information from one or more tick-borne disease-inducing microorganisms selected from the group consisting of bacteria, protozoans, fungi and viruses; contacting a biological sample with the one or more polynucleotide molecules; obtaining hybridization information from the substrate subsequent to contacting the biological sample with the one or more polynucleotide molecules; and relating the hybridization information to information that is related to the tick-borne disease, thereby detecting the tick-born disease at a sensitivity of at least 90% or at a specificity of at least 90%.
  • the biological sample is contacted with the one or more sets of polynucleotide molecules such that at least a portion of the biological sample hybridizes to at least a portion of the one or more sets of
  • the method further comprises sequencing at least a portion of the biological sample.
  • the genomic information from said one or more tick-borne disease-inducing microorganisms comprises sequence information of 16S and 18S ribosomal RNA of the one or more tick-borne disease-inducing microorganisms.
  • said tick- borne disease is detected at a sensitivity of at least 90%> and a specificity of at least 90%>.
  • the disclosure provides for a a microchip, comprising: a substrate; and one or more polynucleotide molecules that are immobilized on or adjacent to the substrate at a plurality of sites, wherein the one or more polynucleotide molecules relate to genomic information from tick-borne disease-inducing microorganisms comprising at least any three of bacteria, protozoans, fungi, and viruses.
  • the bacteria include Borrelia burgdorferi, Anaplasma
  • the protozoans include Babesia microti, Babesia duncani or Toxoplasma gondii.
  • the fungi include Candida spp or Cryptococcus spp.
  • the viruses include tick-borne encephalitis virus or dengue virus, or a virus from the Bunyaviridae family of virses.
  • the genomic information from the tick-borne disease-inducing microorganisms comprises sequence information of 16S and 18S ribosomal RNA of the tick-borne disease-inducing microorganisms.
  • the number of the one or more polynucleotide molecules is at least 60,000.
  • the length of each of the one or more polynucleotide molecules is greater than 70 bp.
  • the length of each of the one or more polynucleotide molecules is less than 70 bp.
  • the tick-borne disease is Lyme Disease.
  • the tick-borne disease-inducing microorganisms comprise bacteria, protozoans and fungi.
  • the tick-borne disease-inducing microorganisms comprise bacteria, protozoans and viruses. In some embodiments, the tick-borne disease-inducing microorganisms comprise bacteria, fungi and viruses. In some embodiments, the tick-borne disease-inducing microorganisms comprise protozoans, fungi and viruses. In some embodiments, the tick-borne disease-inducing microorganisms comprise bacteria, protozoans, fungi and viruses.
  • the disclosure provides for a microchip, comprising: a substrate; and one or more polynucleotide molecules that are immobilized on or adjacent to the substrate at a plurality of sites, wherein the one or more polynucleotide molecules relate to genomic information from tick-borne disease-inducing microorganisms comprising at least any two of bacteria, protozoans, fungi, and viruses, wherein said bacteria are selected from the group consisting of Borrelia burgdorferi,
  • Anaplasma phagocytophilum/Ehrlichia, Rickettsia, Bartonella, Tularemia or Leptospira wherein said protozoans are selected from the group consisting of Babesia microti, Babesia duncani or Toxoplasma gondii, wherein said fungi are selected from the group consisting of Candida spp or Cryptococcus spp, and wherein said viruses are selected from the group consisting of tick-borne encephalitis virus or dengue virus.
  • the tick-borne disease is Lyme Disease.
  • the present disclosure provides for a method of detecting a tick-borne disease, comprising: obtaining a biological sample from a subject, wherein said biological sample comprises one or more nucleic acid molecules; performing pathogen detection analysis on the biological sample; performing host response analysis on the biological sample; and relating a set of information obtained from the pathogen detection analysis and the host response analysis to a set of control information, thereby detecting the tick-borne disease.
  • the biological sample is a blood sample.
  • the method further comprises RNA extraction and DNA extraction from the biological sample.
  • the method further comprises RNA extraction from the peripheral blood mononuclear cells (PBMC) found in the biological sample.
  • PBMC peripheral blood mononuclear cells
  • the pathogen detection analysis and the host response analysis are done simultaneously. In some embodiments, the pathogen detection analysis and the host response analysis are done sequentially. In some embodiments, the pathogen detection analysis is performed by deep sequencing, real time PCR, microarray or using a microchip. In some embodiments, the host response analysis is performed by deep sequencing, real time PCR, microarray or using a microchip. In some embodiments, the information obtained from the host response analysis comprises differential expression level of one or more genes that are related to the tick-borne disease. In some embodiments, the differential expression level of one or more genes is measured for the PBMC found in the biological sample.
  • the microchip comprises one or more polynucleotide molecules that are related to genomic information from one or more tick-borne disease-inducing microorganisms. In some embodiments, the microchip comprises one or more polynucleotide molecules that are related to gene expression information of one or more tick- borne disease-related biomarkers.
  • the pathogen detection analysis allows direct detection of all bacteria species of Borrelia. In some embodiments, the pathogen detection analysis allows direct detection of all virus species of Bunyaviridae. In some embodiments, the subject has acute or chronic tick-borne disease. In some embodiments, no tick-borne related pathogen can be detected in the biological sample.
  • the deep sequencing comprises: obtaining sequence information of said one or more nucleic acid molecules; subtracting human genomic information from the sequence information obtained in (a); and subsequent to subtracting said human genomic information from the sequence information, relating the sequence information to genomic information of one or more pathogens capable of inducing the tick-borne disease.
  • the method further comprises establishing a transcriptome of the biological sample.
  • the one or more genes that are related to the tick-borne disease comprises at least one of IFIT2, IFITM2, SOCS1, SOCS3, STAT1, STAT2, IRF1, TLR1, TLR2, TLR3, TNF, HLA-DQ, HLA-DR, and MYD88.
  • the pathogen detection analysis is based on the sequence information of 16s and 18s ribosomal RNA (e.g., SEQ ID NOs: 57,957-57,958) of one or more pathogens capable of inducing the tick-borne disease.
  • the detecting the tick-borne disease is at a sensitivity of at least 90% and at a specificity of at least 90%>.
  • the disclosure provides for a method of detecting a tick-borne disease at a sensitivity of at least 90%> and a specificity of at least 90%>.
  • the disclosure provides for a method of detecting a tick-borne disease based on the sequence
  • ribosomal RNA e.g., SEQ ID NOs: 57,957-57,958 of one or more pathogens capable of inducing the tick-borne disease at a sensitivity of at least 90%> or at a specificity of at least 90%.
  • FIGURE 1 depicts a method for determining expression levels of tick-borne disease genes in a host.
  • FIGURE 2 demonstrates an example of output from an analysis using the TickChip. Calculated Z
  • FIGURE 3 A depicts a heat map showing a selected cluster of 42 Babesia 18S probes on TickChip vl .0 microarrays corresponding to serial spiked-in dilutions of Babesia microti or Borrelia burgdorferi.
  • the color saturation indicates the normalized magnitude of hybridization intensity on the TickChip.
  • the signature from multiple Babesia 18S probes is highly specific for Babesia microti at dilutions down to 100 genomes/mL (i.e., 1 genome/ml, 1 organism per ml).
  • FIGURE 3B illustrates the measured sensitivity of the TickChip for pathogens spiked into human whole blood and amplified by random amplification or 16S or 18S rRNA PCR
  • FIGURE4 provides a brief, general description of an illustrative and/or suitable example of a
  • FIGURE 5 depicts a method for determining expression levels of tick-borne disease genes in a host.
  • FIGURE 6 depicts a deep-sequencing bioinformatics pipeline for pathogen detection.
  • FIGURE 7 depicts the results from comparing the speed and sensitivity of various bioinformatics tools to process deep sequencing data.
  • FIGURE 8 depicts the analysis of sequences from four different patients, all with acute Lyme disease.
  • FIGURE 9 illustrates that computational challenge various based on the clinical sample type.
  • FIGURE 10 depicts the URPI pipeline for analyzing deep sequencing data.
  • FIGURES 11A-C depict various different pipelines for analyzing deep sequencing data
  • FIGURE 12 depicts the results from comparing the speed of the Sequedex method to other
  • FIGURE 13 depicts the results from comparing the speed of various bioinformatics tools to process deep sequencing data.
  • FIGURE 14 depicts a comparison of Receiver Operating Characteristic (ROC) curves for various methods of mapping deep sequencing data.
  • ROC Receiver Operating Characteristic
  • FIGURE 15 illustrates that SOCS3 expression (a host gene) is increased in patients with Lyme disease relative to control patients.
  • FIGURE 16 illustrates that the expression of multiple host genes is increased in patients with Lyme disease relative to control patients (as detected by deep-sequencing).
  • FIGURES 17A-B depict the use of a Nanostring nCounter Multiplexed Analysis System for a deep- sequencing-based diagnostic assay (without amplification).
  • FIGURE 18 depicts a sample workflow for a deep-sequencing-based diagnostic assay.
  • FIGURE 19 depicts a diagram of a system (e.g., a computer system) for analyzing deep-sequencing data.
  • a system e.g., a computer system
  • FIGURE 20 depicts an overview of one embodiment of the TickChip assay.
  • FIGURE 21 depicts a cluster heat map according to one particular embodiment showing microarray sensitivity to B. burgdorferi using whole blood samples spiked with known concentrations of bacteria and clinical serum samples.
  • FIGURE 22 depicts the positions of particular probes along the B. burgdorferi 16S rRNA gene.
  • FIGURE 23 depicts a cluster heat map according to one particular embodiment showing microarray sensitivity to B. microti using whole blood samples spiked with known concentrations of bacteria and clinical RBC lysate samples.
  • FIGURES 24A-H provide 272 probe sequences based on flaB gene sequences from Borrelia species
  • FIGURES 25A-R provide 616 probe sequences for the detection of tick-borne bunyaviruses (from top to bottom SEQ ID NOs: 58,274-58,889).
  • microchip generally refers to a high -density array of nucleic acid probes
  • microchip can also refer to a "microarray.”
  • nucleotide generally refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • the term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • ATP adenosine triphosphate
  • UDP uridine triphosphate
  • CTP cytosine triphosphate
  • GTP guanosine triphosphate
  • deoxyribonucleoside triphosphates such as dATP, dCTP, d
  • nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled by well known techniques. Labeling can also be carried out with quantum dots.
  • Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine
  • TAMRA 6-carboxy-X-rhodamine
  • ROX 4-(4'dimethylaminophenylazo) benzoic acid
  • DABYL Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid
  • Specific examples of fluorescently labeled nucleotides include [R6G]dUTP,
  • [TAMRA] dUTP [R110]dCTP, [R6G]dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAMJddCTP, [R110]ddCTP, [TAMRA] ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRAJddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be, e.g., biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio- N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-l l-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotide can also refer to "nucleic acid,” (e.g., nucleic acids in a nucleic acid sample), and/or "target nucleic acids.”
  • probe refers to a nucleic acid that is complementary to a nucleotide
  • a probe may be immobilized on a surface of a substrate, where the substrate can have a variety of configurations, e.g., a sheet, bead, or other structure.
  • a probe may be present on a surface of a planar support, e.g., in the form of an array.
  • target nucleic acid generally refers to a nucleic acid to be used in the methods of the disclosure.
  • a target nucleic acid can refer to a chromosomal sequence or an extrachromosomal sequence, (e.g. an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.).
  • a target nucleic acid can be sequenced from a nucleic acid sample.
  • a target nucleic acid can be DNA.
  • a target nucleic acid can herein be used interchangeably with “polynucleotide” and/or "nucleotide sequence”.
  • determining means determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.
  • This disclosure provides for compositions and methods for detecting tick-borne diseases. Detection methods of the present disclosure can be used to diagnose and/or treat a subject suspected of having a disease, such as a tick-bome disease (e.g., Lyme disease, Southern Tick Associated Rash Illness).
  • the method can comprise using microchip methodologies to determine the pathogenic genetic content of a tick-bome disease in a patient sample.
  • FIGURE 1 shows a method for using a microchip to identify genes of tick-bome diseases in a host.
  • a nucleic acid sample can comprise a plurality of nucleic acids 105 can be contacted 110 to a microchip 115.
  • the microchip 115 comprises probes 120. Each probe 120 can comprise a unique sequence that can correspond to a molecular signature of a tick-bome disease and/or patient transcriptome.
  • Each probe 120 can hybridize 125 to a nucleic acid 105.
  • Hybridization can be detected through optical (e.g., fluorescence) detection.
  • hybridization may be detected by electrochemical or electrostatic methods.
  • This disclosure provides for methods for detecting, and sequencing tick-bome diseases.
  • Detection methods of the present disclosure can be used to diagnose and/or treat a subject suspected of having a disease, such as a tick-bome disease (e.g., Lyme disease, Southern Tick Associated Rash Illness).
  • the method can comprise using deep sequencing and data analysis methodologies to determine the pathogenic genetic content of a tick-bome disease in a patient sample.
  • Tick-bome diseases can comprise a plurality of infection stages.
  • Tick-bome diseases can comprise an acute infection stage.
  • a tick-bome disease e.g., Lyme disease, Southern Tick- Associated Rash Illness
  • a tick-bom disease can be described as a spirochetemia phase, wherein the bacteria causing the disease can be detectable in blood.
  • the acute infection stage can be treated with antibiotics in 10-28 days.
  • Tick-bome disease can comprise a chronic infection stage. Chronic infection can lead to neurologic and musculoskeletal disease.
  • Tick-bome diseases can originate from many species.
  • Tick bome-disease can originate from bacteria.
  • Bacteria species can be spirochetes. Bacteria species can include species from the genus Borrelia (e.g., Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii Candidatus Borrelia texasensis, Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borrelia bissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia
  • Borrelia e.g., Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii Candidatus Borrelia texasensis, Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazard
  • Bacteria species can include species from the genus Anaplasma/Ehrlichia (e.g., Anaplasma
  • phagocytophilum phagocytophilum, Ehrlichia phagocytophila, Anaplasma bovis, Anaplasma platys, Anaplasma marginale, Ehrlichia ewingii, Ehrlichia chaffeensis, Ehrlichia canis, Neorickettsia sennetsu).
  • the tick-borne disease can originate from Anaplasma phagocytophilum/Erlichia.
  • Bacteria species can include species from the genus Rickettsia (e.g., Rickettsia aeschlimannii,
  • Bacteria species can include species from the genus Bartonella (e.g., Bartonella alsatica, Bartonella australis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis (also called Stammsii), Bartonella capreoli, Bartonella chomelii, Bartonella clarridgeiae, Bartonella coopersplainsensis, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae, Bartonella japonica, Bartonella koehlerae, Bartonella peromysci, Bartonella phoceensis, Bartonella queenslandernsis, Bartonella quintana, Bartonella rattaustraliani, Bartonella rattimassiliensis, Bartonella rocha
  • Bacteria species can include species that are known to cause Tuleremia (e.g., Francisella tularensis).
  • Bacteria species can include species from the genus Leptospira (e.g, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira alexanderi, Leptospira wellii, Leptospira genomospecies 1 , Leptospira borgpetersenii, Leptospira santarosai, Leptospira kmetyi, Leptospira inadai, Leptospira fainei, Leptospira broomii, Leptospira licerasiae, Leptospira wolffii, Leptospira biflexa, Leptospira meyeri, Leptospira wolbachii, Leptospira genomospecies 3, Leptospira genomospecies 4, Leptospira genom
  • Bacteria species can include species from the genus Treponema (e.g., Treponema amylovorum,
  • Treponema azotonutricium Treponema berlinense, Treponema brennaborense, Treponema bryantii, Treponema caldarium, Treponema denticola, Treponema hyodysenteriae, Treponema innocens, Treponema isoptericolens, Treponema lecithinolyticum, Treponema maltophilum, Treponema medium, Treponema minutum, Treponema pallidum, Treponema paraluiscuniculi, Treponema parvum,
  • Bacteria species can include species from the genus Mycoplasma (e.g., Mycoplasma adleri,
  • Mycoplasma imitans Mycoplasma indiense, Mycoplasma iners, Mycoplasma iowae, Mycoplasma lactucae, Mycoplasma lagogenitalium, Mycoplasma leachii, Mycoplasma leonicaptivi, Mycoplasma leopharyngis, Mycoplasma lipofaciens, Mycoplasma lipophilum, Mycoplasma lucivorax, Mycoplasma luminosum, Mycoplasma maculosum, Mycoplasma melaleucae, Mycoplasma meleagridis, Mycoplasma microti, Mycoplasma moatsii, Mycoplasma mobile, Mycoplasma molare, Mycoplasma mucosicanis, Mycoplasma muris, Mycoplasma mustelae, Mycoplasma mycoides, Mycoplasma mycoides, Mycoplasma mycoides,
  • Mycoplasma mycoides, Mycoplasma neurolyticum, Mycoplasma opalescens, Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, Mycoplasma oxoniensis, Mycoplasma penetrans, Mycoplasma phocicerebrale, Mycoplasma phocidae, Mycoplasma phocirhinis, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma primatum, Mycoplasma pullorum, Mycoplasma pulmonis, Mycoplasma putrefaciens, Mycoplasma salivarium, Mycoplasma simbae, Mycoplasma somnilux, Mycoplasma spermatophilum, Mycoplasma spumans, Mycoplasma sturni, Mycoplasma sualvi, Mycoplasma sualvi, Mycoplasma subdolum, Mycoplasma suis, Mycoplasma synoviae, Mycoplasma testudineu
  • Chlamydia pecorum Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia suis, Chlamydia trachomatis).
  • Tick-borne diseases can originate from protozoans.
  • Prozoans can include species from the gensu
  • Babesia e.g., Babesia bigemina, Babesia bovis, Babesia canis, Babesia cati, Babesia divergens, Babesia duncani, Babesia felis, Babesia gibsoni, Babesia herpailuri, Babesia jakimovi, Babesia major, Babesia microti, Babesia ovate, Babesia pantherae).
  • the tick-borne disease can originate from Babesia microti.
  • the tick-borne disease can originate from Babesia duncani.
  • Prozoans can include species from the genus Toxoplasma (e.g., Toxoplasma gondii). In some
  • tick-borne disease can originate from Toxoplasma gondii.
  • Tick-borne diseases can originate from fungi.
  • Fungi can include species from the genus Candida (e.g., Candida albicans, Candida ascalaphidarum,Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida blattae, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydali, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida lyxosophila, Candida maltosa, Candida marina, Candida membranifaciens, Candida milleri, Candida ole
  • Fungi can include species from the genus Cryptococcus (e.g., Cryptococcus adeliensis, Cryptococcus aerius, Cryptococcus albidosimilis, Cryptococcus antarcticus, Cryptococcus aquaticus, Cryptococcus ater, Cryptococcus bhutanensis, Cryptococcus consortionis, Cryptococcus curvatus, Cryptococcus phenolicus, Cryptococcus skinneri, Cryptococcus terreus, Cryptococcus vishniacci, Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Cryptococcus uniguttulas).
  • Cryptococcus e.g., Cryptococcus adeliensis, Cryptococcus aerius, Cryptococcus albidosimilis, Cryptococcus antarcticus, Cryptococcus aquaticus, Cryptococcus ater, Cryptococcus bhutanensis, Crypto
  • Tick-borne diseases can originate from viruses.
  • Viruses can include tick-borne encephalitis virus and dengue virus.
  • Viruses can include viruses from the Bunyaviridae family of viruses (e.g., hanta
  • Viruses can include the Heartland virus, and the Lone Star virus. Other viruses that may cause or be correlated with tick-borne diseases may include viruses described in, for example, Mahy, Brian W. J. (October 2008). The dictionary of virology. Elsevier. ISBN 978-0-12- 373732-8 which is herein incorporated by reference in its entirety.
  • Affected genes can include IFIT2, IFITM2, suppressor of cytokine signaling, SOCS1, SOCS3, genes in the JAK/STAT pathway, STAT1, STAT2, Interferon type 1, interferon (IFN) induced proteins (e.g., transmembrane protein 2), IFN regulatory factor, IRF1 , IRF7, toll like receptors, TLR1 , TLR2, TLR3, TNF, HLA-DQ, HLA-DR, MYD88, signal transducer and activator of transcription 2, cytokines, NF- kappaB, fibronectin (FN) induced proteins (e.g., with tetratricopeptide).
  • IFIT2 Interferon type 1, interferon (IFN) induced proteins
  • IFN interferon induced proteins
  • IFIT2 or "interferon-induced protein with tetratricopeptide repeats 2" refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%>, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%), or 100%)) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,959; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%), at least about 96%), at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,960.
  • a polypeptide comprising an amino acid sequence having at least about 90% (e.
  • the IFIT2 polypeptide sequence is:
  • the IFIT2 nucleic acid sequence is:
  • IFITM2 or "interferon induced transmembrane protein 2” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,961 ; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,962.
  • the IFITM2 polypeptide sequence is:
  • the IFITM2 nucleic acid sequence is:
  • SOCS1 or "suppressor of cytokine signaling 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,963; and/or (ii) a
  • polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,964.
  • SOCS 1 polypeptide sequence is:
  • MVAHNQ VAADNAVST AAEPRPvRPEP S S S S S SP AAPARPRPCPAVPAP APGDTHFRTFRSHAD YRRITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRDSRQRNCFFALSVKMASGPTSI RVHFQAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRVRPLQELCRQRIVATVGRE NLARIPLNPVLRDYLS SFPFQI (SEQ ID NO: 57,963)
  • the SOCS 1 nucleic acid sequence is:
  • SOCS3 or "suppressor of cytokine signaling 3” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,965; and/or (ii) a
  • polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,966.
  • the SOCS3 polypeptide sequence is:
  • the SOCS3 nucleic acid sequence is:
  • STAT1 or “signal transducer and activator of transcription 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to one of the amino acid sequences set forth in SEQ ID NO: 57,967 and 57,968; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to one of the nucleic acid sequences set forth in SEQ ID NO: 57,969 and 57,970.
  • STAT1 (isoform alpha) polypeptide sequence is:
  • STAT1 (isoform beta) polypeptide sequence is:
  • STAT1 isoform alpha nucleic acid sequence
  • STAT1 (isoform beta) nucleic acid sequence is:
  • STAT2 or “signal transducer and activator of transcription 2” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to one of the amino acid sequences set forth in SEQ ID NO: 57,971 and 57,972; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to one of the nucleic acid sequences set forth in SEQ ID NO: 57,973 and 57,974.
  • STAT2 (isoform 1) polypeptide sequence is:
  • STAT2 (isoform 2) polypeptide sequence is:
  • STAT2 (isoform 1) nucleic acid sequence is:
  • STAT2 (isoform 2) nucleic acid sequence is:
  • IRF1 or "interferon regulatory factor 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,975; and/or (ii) a
  • polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,976.
  • the IRF1 polypeptide sequence is:
  • the IRFl nucleic acid sequence is:
  • TLR1 or "toll-like receptor 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,977; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,978.
  • a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%,
  • TLR1 polypeptide sequence is:
  • TLR1 nucleic acid sequence is:
  • TLR2 or "toll-like receptor 2” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,979; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,980. [00117] The TLR2 polypeptide sequence is:
  • TLR2 nucleic acid sequence is:
  • TLR3 or "toll-like receptor 3” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,981 ; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,982.
  • TLR3 polypeptide sequence is:
  • TLR3 nucleic acid sequence is:
  • TNF tumor necrosis factor
  • a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,983; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,984.
  • TNF polypeptide sequence is:
  • TNF nucleic acid sequence is:
  • HLA-DQ or "major histocompatibility complex, class II, DQ” encompasses HLA-DQAl (major histocompatibility complex, class II, DQ alpha 1) and HLA-DQB 1 (major histocompatibility complex, class II, DQ beta 1).
  • HLA-DQAl or “major histocompatibility complex, class II, DQ alpha 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%>, at least about 99.5%), or 100%)) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 57,985; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90%> (e.g., at least about 92%>, at least about 95%>, at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%>, at least about 99.5%>, or 100%) nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57,986.
  • HLA-DQAl polypeptide sequence is:
  • HLA-DQAl nucleic acid sequence is:
  • HLA-DQB 1 or "major histocompatibility complex, class II, DQ beta 1” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%>, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%), or 100%)) amino acid sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 57,987 and 57,988; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90%) (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%), at least about 98%), at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to one of the nucleic acid sequences set forth in SEQ ID NOs: 57,
  • HLA-DQB 1 (isoform 1) polypeptide sequence is:
  • HLA-DQB 1 (isoform 2) polypeptide sequence is:
  • HLA-DQB 1 (variant 1) nucleic acid sequence is:
  • AAAAAAAAAAAAAAA SEQ ID NO: 57,989
  • the HLA-DQB1 (variant 2) nucleic acid sequence is:
  • the HLA-DQB1 (variant 3) nucleic acid sequence is:
  • MYD88 or "myeloid differentiation primary response 88” refers to (i) a polypeptide comprising an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 57,992- 57,996; and/or (ii) a polynucleotide comprising a nucleotide sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) nucleic acid sequence identity to one of the nucleic acid sequences set forth in SEQ ID NOs: 57,997-58,001.
  • the MYD88 (isoform 1) polypeptide sequence is:
  • the MYD88 (isoform 2) polypeptide sequence is:
  • the MYD88 (isoform 3) polypeptide sequence is:
  • the MYD88 (isoform 4) polypeptide sequence is: [00156] MRPDRAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEED CQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGAAGWWWLSLMITCRARNVT SRPNLHSASLQVPIRSD (SEQ ID NO: 57,995)
  • the MYD88 (isoform 5) polypeptide sequence is:
  • the MYD88 (isoform 1) nucleic acid sequence is:
  • the MYD88 (isoform 2) nucleic acid sequence is:
  • the MYD88 (isoform 3) nucleic acid sequence is:
  • the MYD88 (isoform 4) nucleic acid sequence is:
  • the MYD88 (isoform 5) nucleic acid sequence is:
  • HLA-DR or “major histocompatibility complex, class II, DR” encompasses HLA-DRA (major
  • accession numbers for protein and nucleic acid sequences for HLA-DRA include: NP 061984.2 and NM 019111.4.
  • accession numbers for nucleic acid and protein sequences for HLA-DRB include: NM 001243965.1, NM 002124.3, NP 002115.2, and NP_001230894.1 (beta 1); NM_022555.3 and NP_072049.2 (beta 3); NM_021983.4 and
  • NP_068818.4 (beta 4); and NM_002125.3 and NP 002116.2 (beta 5).
  • Nucleic acids in a nucleic acid sample can be derived from a variety of sources including any species containing genetic material.
  • a nucleic acid sample can be derived from human, mammal, non-human mammal, ape, monkey, chimpanzee, reptilian, amphibian, avian, insect or various invertebrate sources.
  • a nucleic acid sample can be derived from microorganisms which can include, but are not limited to, unicellular organisms or multi-cellular organisms, bacteria, parasites, fungi, protists, algae, larvae, nematodes, worms, viruses and any combination thereof.
  • a nucleic acid sample can be extracted from variety of tissues and tissue types.
  • a nucleic acid sample can be fetal in origin (e.g., fluid taken from a pregnant subject), or can be derived from tissue of the subject itself.
  • a nucleic acid sample can be found as cell- free, or in a state not contained within cells.
  • a nucleic acid sample can be extracted from, for example, a bodily fluid or tissue.
  • a nucleic acid sample can originate from bodily fluids, dissociated tumor specimens, cultured cells, and any combination thereof.
  • a nucleic acid sample can come from one or more individuals.
  • One or more nucleic acid samples can come from the same individual. One non limiting example would be if one nucleic acid sample came from an individual's blood and a second nucleic acid sample came from an individual's tumor biopsy.
  • sources of nucleic acid samples can include but are not limited to, blood, serum, plasma, nasal swab or nasopharyngeal wash, saliva, urine, gastric fluid, spinal fluid, tears, stool, mucus, sweat, earwax, oil, glandular secretion, cerebral spinal fluid, tissue, semen, vaginal fluid, interstitial fluids, including interstitial fluids derived from tumor tissue, ocular fluids, spinal fluid, throat swab, breath, hair, finger nails, skin, biopsy, placental fluid, amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum, pus, micropiota, meconium, breast milk and/or other excretions.
  • a source of a nucleic acid sample can comprise blood.
  • a source of a nucleic acid sample can comprise pheripheral blood mononuclear cells (PBMCs).
  • PBMCs pheripheral blood mononuclear cells
  • a source of a nucleic acid sample can be a tissue sample.
  • Tissue samples can include, but are not limited to, connective tissue, muscle tissue, nervous tissue, epithelial tissue, cartilage, cancerous or tumor sample, or bone.
  • the tissue sample can be provided from a human or animal.
  • the tissue sample can be provided from a mammal, vertebrate, invertebrate, ticks, bacteria, protozoa, fungi, and viruses.
  • the tissue sample can be collected from a living or dead organism.
  • the tissue sample can be collected fresh from an organism or can have undergone some form of pre-processing, storage, or transport.
  • a nucleic acid can be isolated from a nucleic acid sample by techniques such as nucleic extraction using common techniques such as cell lysis, removing cell membranes, removing proteins, removing RNA, and precipitating the DNA.
  • Cell lysis can be performed using chaotropic salts (e.g., guanidinium isothiocyanate and ammonium isothiocyanate in high concentration).
  • chaotropic salts e.g., guanidinium isothiocyanate and ammonium isothiocyanate in high concentration.
  • Removal of proteins can be performed with detergents and/or surfactants (e.g., SDS, CHAPS).
  • Removal of RNA can be performed with RNases (e.g., RNase A).
  • DNA can be precipitated with ethanol and/or isopropanol.
  • RNA extraction can be performed largely similarly to DNA extraction.
  • RNA can be extracted with a reagent comprising phenol-chloroform and/or a chaotropic denaturing agent (e.g., guanidinium thiocyanate).
  • RNA can be precipitated with sodium acetate.
  • RNA extraction can be performed from a whole blood sample. Extracted RNA can be purified by a purification agent that binds to a RNA's polyadenylated tail.
  • the present disclosure provides an array of nucleic acid probes.
  • a probe may be present on a surface of a planar support, e.g., in the form of an array.
  • the present disclosure provides a microchip having immobilized thereon a subject nucleic acid probe array.
  • an array of surface-bound polynucleotides (probes) is not a mixture of surface-bound polynucleotides because the species of surface-bound (immobilized) polynucleotides are spatially distinct and the array is addressable.
  • a subject array comprises 1, 10, 100, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, or more probes.
  • a subject array can comprise from about 10 to about 10 , from about 10 to about 10 , from about 10 to about 10 5 different nucleic acid probes.
  • a subject array comprises from 5 to 10, from 10 to 50, from 50 to 100, from 10 2 to 2 x 10 2 , from 2 x 10 2 to 5 x 10 2 , from 5 x 10 2 to 10 3 , from 10 3 to 5 x 10 3 , from 5 x 10 3 to 10 4 , from 5 x 10 4 to the complete set of probes having nucleotide sequences set forth in SEQ ID NOs:3-57,956.
  • a subject array can include nucleic acid probes that hybridize to a Borrelia spp. nucleic acid.
  • a subject array can include nucleic acid probes comprising nucleotide sequences set forth in SEQ ID NOs: 10, 12, 13, 15, etc.
  • a subject array can comprise nucleic acid probes that hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, from 5 to 10, from 10 to 20, or more than 20, different tick-borne pathogens.
  • Probes can be can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 nucleotides (nt) or more in length.
  • a probe in a subject array has a length of from about 50 nt to about 100 nt, e.g., about 70 nt.
  • RT-PCR also referred to as quantitative -PCR (QPCR)
  • QPCR quantitative -PCR
  • RT-PCR can detect an amount of amplifiable nucleic acid present in a sample.
  • QPCR can be used to identify genes that can cause or can be correlated to a tick-borne disease.
  • QPCR is a technique based on the polymerase chain reaction, and can be used to amplify and simultaneously quantify a target nucleic acid.
  • QPCR can allow for both detection and quantification of a specific sequence in a DNA sample.
  • the procedure can follow the general principle of polymerase chain reaction, with the additional feature that the amplified DNA can be quantified as it accumulates in the reaction in real time after each amplification cycle.
  • Two methods of quantification can be: (1) use of fluorescent dyes that intercalate with double-stranded DNA, and (2) modified DNA oligonucleotide probes that fluoresce when hybridized with a
  • a DNA-binding dye can bind to all double-stranded (ds)DNA in PCR, resulting in fluorescence of the dye.
  • ds double-stranded
  • An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and can be measured at each cycle, thus allowing DNA
  • the reaction can be prepared similarly to a standard PCR reaction, with the addition of fluorescent (ds)DNA dye.
  • the reaction can be run in a thermocycler, and after each cycle, the levels of fluorescence can be measured with a detector; the dye can only fluoresce when bound to the (ds)DNA (i.e., the PCR product).
  • the (ds)DNA concentration in the PCR can be determined. The values obtained do not have absolute units associated with it.
  • a comparison of a measured DNA/RNA sample to a standard dilution can give a fraction or ratio of the sample relative to the standard, allowing relative comparisons between different tissues or experimental conditions.
  • the expression of a target gene can be normalized to a stably expressed gene. This can allow for correction of possible differences in nucleic acid quantity or quality across samples.
  • the second method can use a sequence-specific RNA or DNA-based probe to quantify only the DNA containing the probe sequence; therefore, use of the reporter probe can increase specificity, and can allow quantification even in the presence of some non-specific DNA amplification. This can allow for multiplexing, (i.e., assaying for several genes in the same reaction by using specific probes with differently colored labels), provided that all genes are amplified with similar efficiency.
  • This method can be carried out with a DNA-based probe with a fluorescent reporter (e.g.
  • 6-carboxyfluorescein at one end and a quencher (e.g., 6-carboxy- tetramethylrhodamine) of fluorescence at the opposite end of the probe.
  • a quencher e.g., 6-carboxy- tetramethylrhodamine
  • the close proximity of the reporter to the quencher can prevent detection of its fluorescence.
  • Breakdown of the probe by the 5' to 3' exonuclease activity of a polymerase e.g., Taq polymerase
  • An increase in the product targeted by the reporter probe at each PCR cycle can result in a proportional increase in fluorescence due to breakdown of the probe and release of the reporter
  • the reaction can be prepared similarly to a standard PCR reaction, and the reporter probe can be added.
  • both probe and primers can anneal to the DNA target.
  • Polymerization of a new DNA strand can be initiated from the primers, and once the polymerase reaches the probe, its 5 '-3 '-exonuclease can degrade the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
  • Fluorescence can be detected and measured in a real-time PCR thermocycler, and geometric increase of fluorescence can correspond to exponential increase of the product is used to determine the threshold cycle in each reaction.
  • Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line).
  • a threshold for detection of fluorescence above background can be determined.
  • Amounts of nucleic acid e.g., RNA or DNA
  • Amounts of nucleic acid can be determined by comparing the results to a standard curve produced by a real-time PCR of serial dilutions (e.g.
  • the QPCR reaction can involve a dual fluorophore approach that takes advantage of fluorescence resonance energy transfer (FRET), (e.g., LIGHTCYCLER hybridization probes, where two oligonucleotide probes can anneal to the amplicon).
  • FRET fluorescence resonance energy transfer
  • the oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer.
  • labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: SCORPIONS probes, Sunrise (or AMPLIFLOUR) primers, and LUX primers and MOLECULAR BEACONS probes.
  • the QPCR reaction can use fluorescent Taqman methodology and an instrument capable of measuring fluorescence in real time (e.g., ABI Prism 7700 Sequence Detector).
  • the Taqman reaction can use a hybridization probe labeled with two different fluorescent dyes. One dye can be a reporter dye (6-carboxyfluorescein), the other can be a quenching dye (6-carboxy-tetramethylrhodamine).
  • the fluorescent hybridization probe can be cleaved by the 5 '-3' nucleolytic activity of the DNA polymerase. On cleavage of the probe, the reporter dye emission can no longer transferred efficiently to the quenching dye, resulting in an increase of the reporter dye fluorescent emission spectra.
  • Any nucleic acid quantification method including real-time methods or single-point detection methods may be use to quantify the amount of nucleic acid in the sample. The detection can be performed several different methodologies (e.g., staining, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection;
  • the quantification may or may not include an amplification step.
  • the quantitation may not be experimental.
  • Microchips can be used for determining the expression level of a plurality of genes in a nucleic acid sample. For example, a microchip can be used to identify relative expression levels of genes causing or correlated to a tick-borne disease. Microchips can be used for determining sequence identity of a plurality of sequences in a nucleic acid sample.
  • a microchip can comprise a substrate.
  • Substrates can include, but are not limited to, glass and
  • plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, and plastics.
  • Microchips can comprise a plurality of polynucleotide probes.
  • a microchip can comprise about 1, 10, 100, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 or more probes.
  • a subject microcip can comprise from about 10 to about 10 2 , from about 10 2 to about 10 3 , from about 10 3 to about 10 4 , from about 10 4 to about 10 5 different nucleic acid probes.
  • Probes on the microchip can be can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 nucleotides or more in length.
  • a subject microchip comprises from 5 to 10, from 10 to 50, from 50 to 100, from 10 2 to 2 x 10 2 , from 2 x 10 2 to 5 x 10 2 , from 5 x 10 2 to 10 3 , from 10 3 to 5 x 10 3 , from 5 x 10 3 to 10 4 , from 5 x 10 4 to the complete set of probes having nucleotide sequences set forth in SEQ ID NOs:3-57,956.
  • a subject microchip can include nucleic acid probes that hybridize to a Borrelia spp. nucleic acid.
  • a subject microchip can include nucleic acid probes comprising nucleotide sequences set forth in SEQ ID NOs: 10, 12, 13, 15, etc.
  • a subject array can comprise nucleic acid probes that hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, from 5 to 10, from 10 to 20, or more than 20, different tick-borne pathogens.
  • Probes can be can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 nucleotides (nt) or more in length.
  • a probe in a subject array has a length of from about 50 nt to about 100 nt, e.g., about 70 nt.
  • probes can comprise sequence information for a specific set of genes and/or species.
  • a microchip can comprise probes that are complementary to nucleic acid sequences in bacteria, protozoans, fungi, and/or viruses.
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Borrelia species (e.g., Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii Candidatus Borrelia texasensis, Borrelia afzelii, Borrelia americana, Borrelia andersonii, Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borrelia bissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia californiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Bor
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Anaplasma/Ehrlichia species (e.g., Anaplasma phagocytophilum, Ehrlichia phagocytophila, Anaplasma bovis, Anaplasma platys, Anaplasma marginale, Ehrlichia ewingii, Ehrlichia chaffeensis, Ehrlichia canis, Neorickettsia sennetsu).
  • the tick-borne disease can originate from Anaplasma phagocytophilum/Erlichia.
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Rickettsia species (e.g., Rickettsia aeschlimannii, Rickettsia africae, Rickettsia akari, Rickettsia asiatica, Rickettsia australis, Rickettsia canadensis, Rickettsia conorii, Rickettsia cooleyi, Rickettsia felis, Rickettsia heilongjiangensis, Rickettsia helvetica, Rickettsia honei, Rickettsia hulinii, Rickettsia japonica, Rickettsia massiliae, Rickettsia montanensis, Rickettsia parked, Rickettsia peacock
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Bartonella species (e.g., Bartonella alsatica, Bartonella australis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis (also called Stammsii), Bartonella capreoli, Bartonella chomelii, Bartonella clarridgeiae, Bartonella coopersplainsensis, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae, Bartonella japonica, Bartonella koehlerae, Bartonella peromysci, Bartonella phoceensis, Bartonella queenslandernsis, Bartonella quintana, Bartonella rattaustraliani, Bartonella rat
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of bacteria that is known to cause Tuleremia (e.g., Francisella tularensis).
  • Tuleremia e.g., Francisella tularensis
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Leptospira (e.g, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira alexanderi, Leptospira wellii, Leptospira genomospecies 1 , Leptospira borgpetersenii, Leptospira santarosai, Leptospira kmetyi, Leptospira inadai, Leptospira fainei, Leptospira broomii, Leptospira licerasiae, Leptospira wolffii, Leptospira biflexa, Leptospira meyeri, Leptospira wolbachii, Leptospira genomospecies 3, Leptospira genomo
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Babesia (e.g., Babesia bigemina, Babesia bovis, Babesia canis, Babesia cati, Babesia divergens, Babesia duncani, Babesia felis, Babesia gibsoni, Babesia herpailuri, Babesia jakimovi, Babesia major, Babesia microti, Babesia ovate, Babesia pantherae).
  • the tick-borne disease can originate from Babesia microti.
  • the tick-borne disease can originate from Babesia duncani.
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome Toxoplasma (e.g., Toxoplasma gondii).
  • Toxoplasma gondii a nucleic acid sequence in the genome Toxoplasma
  • the tick-borne disease can originate from Toxoplasma gondii.
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Candida (e.g., Candida albicans, Candida ascalaphidarum,Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida blattae, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydali, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida lyxosophila, Candida maltosa, Candida marina, Candida membranifaciens, Candida milleri, Candida ole
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of Cryptococcus (e.g., Cryptococcus adeliensis, Cryptococcus aerius, Cryptococcus albidosimilis, Cryptococcus antarcticus, Cryptococcus aquaticus, Cryptococcus ater, Cryptococcus bhutanensis, Cryptococcus consortionis, Cryptococcus curvatus, Cryptococcus phenolicus, Cryptococcus skinneri, Cryptococcus terreus, Cryptococcus vishniacci, Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus albidus, Cryptococcus uniguttulas).
  • Cryptococcus e.g., Cryptococcus adeliensis, Cryptococcus aerius, Cryptococcus albidosimilis, Cryptococcus antarcticus, Cryptococcus aquaticus, Cryptococcus ater, Crypto
  • a probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome of a virus (e.g., tick-borne encephalitis virus and dengue virus and other viruses that may cause or be correlated with tick-borne diseases may include viruses described in, for example, Mahy, Brian W. J. (October 2008). The dictionary of virology. Elsevier. ISBN 978-0-12-373732-8 which is herein incorporated by reference in its entirety.)
  • a virus e.g., tick-borne encephalitis virus and dengue virus and other viruses that may cause or be correlated with tick-borne diseases may include viruses described in, for example, Mahy, Brian W. J. (October 2008). The dictionary of virology. Elsevier. ISBN 978-0-12-373732-8 which is herein incorporated by reference in its entirety.
  • a probe can be complementary to a nucleic acid sequence encoding a host protein.
  • a probe can be complementary to a non-coding nucleic acid sequence.
  • a probe can be complementary to a DNA sequence.
  • a probe can be complementary to an RNA sequence.
  • a probe can be complementary to a sequence of 16S and/or 18S ribosomal RNA.
  • a probe can be complementary to a sequence of 16S and/or 18S ribosomal DNA.
  • a probe can be complementary to a sequence of bacterial ribosomal DNA.
  • a probe can be complementary to a sequence of viral DNA.
  • a probe can be complementary to a sequence of bacterial ribosomal RNA and/or rriRNA.
  • a probe can be complementary to a sequence of viral RNA.
  • Probes can be immobilized on a microchip. The immobilization of polynucleotides on a solid
  • substrate can be achieved by direct synthesis (e.g., photolithographic synthesis) of polynucleotides on a solid substrate or by immobilization (spotting) of previously synthesized polynucleotides on predetermined regions of a solid substrate.
  • Polynucleotides can be immobilized on a microarray substrate by activating a surface of a solid substrate with a nucleophilic functional group (e.g., an amino group), coupling biomolecules (e.g., polynucleotides) activated with a good leaving group to the surface-activated solid substrate, and removing unreacted reactants.
  • Probes can be immobilized to a bead further conjugated through a covalent or ionic attachment to a solid support.
  • Probes can be immobilized onto a substrate using a specific film having a low conductivity and a low melting temperature, namely a gold film.
  • An applied electromagnetic radiation can melt and can ablate the film at the impingement site.
  • the film can be in contact with a colloidal dispersion and upon melting can generate a convective flow at the reaction site, thereby leading to adhering of an insoluble particle in the dispersion to the specifically melted site.
  • a microchip can analyze a nucleic acid sample comprising nucleic acids of unknown identity (e.g., test sample) by comparing the nucleic acid sample of unknown identity with a reference sample.
  • a nucleic acid sample can be prepared from DNA (e.g., isolated DNA, genomic DNA,
  • a nucleic acid sample can be prepared from RNA.
  • RNA can be reverse transcribed into DNA with a gene-specific primer or a univerisal primer.
  • the reverse transcribed DNA e.g., cDNA
  • Rnase or base e.g., NaOH
  • the cDNA can be labelled with a dye (e.g, Cy3, Cy5) with N-hydroxysuccinimide chemistry or similar labeling chemistries.
  • Suitable fluorescent dyes can include a variety of commercial dyes and dye derivatives such as those that are denoted Alexa, Fluorescein, Rhodamine, FAM, TAMRA, Joe, ROX, Texas Red, BODIPY, FITC, Oregon Green, Lissamine and others.
  • the reference sample can be labeled with a different dye than the test sample.
  • test sample and the reference sample can be applied to a microchip to contact multiple spots
  • the test sample and the reference sample can be applied to the microchip under hybridizing conditions that allow the nucleic acids in the nucleic acid sample to bind to a complement probe on the microchip.
  • Various reaction steps may be performed with the bound molecules in the microchip, including exposure of bound reactant molecules to washing steps. The progress or outcome of the reaction may be monitored at each spot (e.g., probe) in the microchip in order to characterize the nucleic acid sample immobilized on the chip.
  • Microchip analysis usually requires an incubation period that can range from minutes to hours. The duration of the incubation period can be assay dependent and can be determined by a variety of factors, such as the type of reactant, degree of mixing, sample volume, target copy number, and density of the array.
  • nucleic acids in the nucleic acid sample can be in intimate contact with the microchip probes.
  • Detection can be performed using a confocal scanning instrument with laser excitation and
  • photomultiplier tube detection such as the ScanArray 3000 provided by GSI Lumonics (Bellerica, MA).
  • Confocal and non-confocal fluorescent detection systems can be used to implement the method such as those provided by Axon Instruments (Foster City, CA), Genetic MicroSystems (Santa Clara, CA), Molecular Dynamics (Sunnyvale, CA) and Virtek (Woburn, MA).
  • Alternative detection systems can include scanning systems that use gas, diode and solid state lasers as well as those that use a variety of other types of illumination sources such as xenon and halogen bulbs.
  • detectors can include cameras that use charge coupled device (CCD) and complementary metal oxide silicon (CMOS) chips.
  • CCD charge coupled device
  • CMOS complementary metal oxide silicon
  • the ratio of the intensities of the two dyes from the test sample and the reference sample can be any ratio of the intensities of the two dyes from the test sample and the reference sample.
  • the strength of the signal detected from a given microchip spot can be directly proportional to the degree of hybridization of a nucleic acid in the sample to the probe at a given spot (e.g., a spot comprises a probe).
  • Analysis of the fluorescence intensities of hybridized microchips can include spotsegmentation, background determination (and possible subtraction), elimination of bad spots, followed by a method of normalization to correct for any remaining noise. Normalization techniques can include global normalization on all spots or a subset of the spots such as housekeeping genes, prelog shifting to obtain better baseline matches, or in the case of two (or more) channel hybridizations finding the best fit that helps to give an M vs.
  • a plot that is centered about M 0 and/or that helps to give a log(Red) vs. log( Green) plot that is centered about the diagonal with the smallest spread.
  • Scaling, shifting, best fits through scatter plots, etc. can be techniques utilized to normalize microarray datasets and to give better footing for subsequent analysis. Most of these normalization methods have some underlying hypothesis behind them (such as "most genes within the study do not vary much"). These methods can determine the relative expression levels of genes originating from a tick (e.g., related to atick-borne disease). Deep Sequencing
  • Deep sequencing can use techniques in Next Generation Sequencing (NGS). Deep sequencing
  • Deep sequencing techniques can identify host gene responses to infection with a tick-borne disease and/or identify genomic content originating from a tick-borne disease. Identification of the genomic content originating from at tick-borne disease can be used to diagnose a patient and/or treat the disease. Deep sequencing techniques can demonstrate uniform coverage genome at over 10,000 fold, for example. The high coverage can allow for the detection of nucleotide changes. Moreover, deep sequencing can simultaneously detect small indels and large deletions, map exact breakpoints, and monitor copy number changes.
  • a nucleic acid sample to be used in deep sequencing can be isolated and fragmented. Fragmentation can be performed through physical, mechanical or enzymatic methods. Physical fragmentation can include exposing a target nucleic acid to heat or to ultraviolet (UV) light. Mechanical disruption can be used to mechanically shear a target nucleic acid into fragments of the desired range. Mechanical shearing can be accomplished through a number of methods, including repetitive pipetting of the nucleic acid, sonication and nebulization. Nucleic acids in a nucleic acid sample can also be fragmented using enzymatic methods. In some cases, enzymatic digestion can be performed using enzymes such as using restriction enzymes.
  • Restriction enzymes can be used to perform specific or non-specific fragmentation of nucleic acids.
  • the methods can use one or more types of restriction enzymes, generally described as Type I enzymes, Type II enzymes, and/or Type III enzymes.
  • Type II and Type III enzymes can recognize specific sequences of nucleotides within a target nucleic acid (a "recognition sequence” or "recognition site”). Upon binding and recognition of these sequences, Type II and Type III enzymes can cleave the target nucleic acid sequence.
  • cleavage can result in a target nucleic acid fragment with a portion of overhanging single stranded DNA, called a "sticky end.” In other cases, cleavage will not result in a fragment with an overhang, creating a "blunt end.”
  • the methods can comprise use of restriction enzymes that generate either sticky ends or blunt ends. Sticky and/or blunt ends can be 5' phosphorylated using commercial kits, such as those available from Epicentre
  • an adapter can comprise any oligonucleotide having a sequence, at least a portion of which is known, that can be joined to a target nucleic acid.
  • An adapter can comprise an identifier sequence (a "barcode") that is a distinct sequence for each target nucleic acid in the nucleic acid sample.
  • Adapter oligonucleotides can comprise DNA, RNA, nucleotide analogues, non-canonical nucleotides, labeled nucleotides, modified nucleotides, or combinations thereof.
  • Adapter oligonucleotides can be single-stranded, double-stranded, or partial duplex.
  • a partial-duplex adapter can comprise one or more single-stranded regions and one or more double-stranded regions.
  • Double-stranded adapters can comprise two separate oligonucleotides hybridized to one another (also referred to as an "oligonucleotide duplex"), and hybridization can leave one or more blunt ends, one or more 3' overhangs, one or more 5' overhangs, one or more bulges resulting from mismatched and/or unpaired nucleotides, or any combination of these.
  • a single-stranded adapter can comprises two or more sequences that can
  • hybridization can yield a hairpin structure (hairpin adapter).
  • hairpin adapter When two hybridized regions of an adapter are separated from one another by a non-hybridized region, a "bubble" structure can result.
  • Adapters comprising a bubble structure can consist of a single adapter oligonucleotide comprising internal hybridizations, or may comprise two or more adapter oligonucleotides hybridized to one another. Internal sequence hybridization, such as between two hybridizable sequences in an adapter, can produce a double-stranded structure in a single-stranded adapter oligonucleotide.
  • Adapters of different kinds can be used in combination, such as a hairpin adapter and a double-stranded adapter, or adapters of different sequences. Adapters can be those corresponding to commercially available sequencing platforms.
  • Adapters can be manipulated prior to combining with target nucleic acids. For example, terminal
  • Adapters can contain one or more of a variety of sequence elements, including but not limited to, one or more amplification primer annealing sequences or complements thereof, one or more sequencing primer annealing sequences or complements thereof, one or more barcode sequences, one or more common sequences shared among multiple different adapters or subsets of different adapters, one or more restriction enzyme recognition sites, one or more overhangs complementary to one or more target polynucleotide overhangs, one or more probe binding sites (e.g. for attachment to a sequencing platform, such as a flow cell for massive parallel sequencing, such as developed by Illumina, Inc.), one or more random or near-random sequences (e.g.
  • Adapters can be ligated to target nucleic acids. Ligation can refer to the covalent attachment of two separate polynucleotides to produce a single larger polynucleotide with a contiguous backbone.
  • Methods for joining two polynucleotides can include without limitation, enzymatic and non-enzymatic (e.g. chemical) methods.
  • An adapter oligonucleotide can be joined to a target polynucleotide by a ligase, for example a DNA ligase or RNA ligase.
  • ligases each having characterized reaction conditions can include, without limitation NAD+ -dependent ligases including tRNA ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia coli DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), thermostable ligase, Ampligase thermostable DNA ligase, VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, and novel ligases discovered by bioprospecting; ATP-dependent ligases including T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase IV, and novel ligases discovered by bioprospecting; and wild-type, mutant isoforms, and genetically engineered variants thereof.
  • a 5' phosphate can be utilized in a ligation reaction.
  • the 5' phosphate can be provided by the target polynucleotide, the adapter oligonucleotide, or both.
  • 5' phosphates can be added to or removed from polynucleotides to be joined, as needed.
  • Sequence determination can be performed using commercially available deep sequencing technologies that determine many (typically thousands to billions) nucleic acid sequences in an intrinsically parallel manner, where many sequences can be read out preferably in parallel using a high throughput serial process.
  • Such methods include but are not limited to pyrosequencing (for example, as commercialized by 454 Life Sciences, Inc., Branford, Conn.), sequencing by ligation (for example, as commercialized in the SOLiDTM technology, Life Technology, Inc., Carlsbad, Calif), sequencing by synthesis using modified nucleotides (such as commercialized in TruSeqTM and HiSeqTM technology by Illumina, Inc., San Diego, Calif, HeliScopeTM by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, Calif), sequencing by ion detection
  • a sequencing technique that can be used in the methods of the provided invention can include, for example, Helicos True Single Molecule Sequencing (tSMS).
  • tSMS Helicos True Single Molecule Sequencing
  • a DNA sample can be cleaved into strands of approximately 100 to 200 nucleotides, and a polyA sequence can be added to the 3' end of each DNA strand.
  • Each strand can be labeled by the addition of a fluorescently labeled adenosine nucleotide.
  • the DNA strands can be hybridized to a flow cell, which can contain millions of oligo-T capture sites that can be immobilized to the flow cell surface.
  • the templates can be at a density of about 100 million templates/cm2 .
  • the flow cell can be loaded into an instrument, (e.g., HeliScope.TM.) sequencer, and a laser can illuminate the surface of the flow cell, revealing the position of each template.
  • a CCD camera can map the position of the templates on the flow cell surface.
  • the template fluorescent label can be cleaved and washed away.
  • the sequencing reaction can begin by introducing a DNA polymerase and a fluorescently labeled nucleotide.
  • the oligo-T nucleic acid can serve as a primer.
  • the polymerase can incorporate the labeled nucleotides to the primer in a template directed manner.
  • the templates that have directed incorporation of the fluorescently labeled nucleotide can be detected by imaging the flow cell surface. After imaging, a cleavage step can removes the fluorescent label, and the process can be repeated with other fluorescently labeled nucleotides until the desired read length is achieved. Sequence information can be collected with each nucleotide addition step.
  • DNA sequencing technique that can be used in the methods of the provided invention is 454 sequencing (Roche) 454 sequencing can involve two steps. In the first step, DNA can be sheared into fragments of approximately 300-800 base pairs, and the fragments can be blunt ended. Oligonucleotide adapters can be ligated to the ends of the fragments. The adapters can serve as primers for amplification and sequencing of the fragments. The fragments can be attached to DNA capture beads, (e.g.,streptavidin-coated beads using, e.g., Adapter B, which contains 5'-biotin tag). The fragments attached to the beads can be PCR amplified within droplets of an oil-water emulsion.
  • DNA capture beads e.g.,streptavidin-coated beads using, e.g., Adapter B, which contains 5'-biotin tag.
  • the result can be multiple copies of clonally amplified DNA fragments on each bead.
  • the beads can be captured in wells (pico-liter sized). Pyrosequencing can be performed on each DNA fragment in parallel. Addition of one or more nucleotides can generate a light signal that can be recorded by a CCD camera in a sequencing instrument. The signal strength can be proportional to the number of nucleotides incorporated. Pyrosequencing can make use of pyrophosphate (PPi) which can be released upon nucleotide addition. PPi can be converted to ATP by ATP sulfurylase in the presence of adenosine 5' phosphosulfate. Luciferase can use ATP to convert luciferin to oxyluciferin, and this reaction can generate light that can be detected and analyzed.
  • PPi pyrophosphate
  • Luciferase can use ATP to convert luciferin to oxyluciferin, and this reaction can generate light that can be detected and analyzed.
  • SOLiD sequencing nucleic acids in a nucleic acid sample can be sheared into fragments, and adapters can be attached to the 5' and 3' ends of the fragments to generate a fragment library.
  • internal adapters can be introduced by ligating adapters to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragment to generate an internal adapter, and attaching adapters to the 5' and 3' ends of the resulting fragments to generate a mate -paired library.
  • the templates can be denatured and beads can be enriched to separate the beads with extended templates.
  • Templates on the selected beads can be subjected to a 3' modification that permits bonding to a glass slide.
  • the sequence can be determined by sequential hybridization and ligation of partially random oligonucleotides with a central determined base (or pair of bases) that can be identified by a specific fluorophore. After a color is recorded, the ligated oligonucleotide can be cleaved and removed and the process can then be repeated.
  • Ion Torrent sequencing Another example of a DNA sequencing technique that can be used in the methods of the provided invention is Ion Torrent sequencing. In Ion Torrent sequencing, nucleic acids in a nucleic acid sample can be sheared into fragments of approximately 300-800 base pairs, and the fragments can be blunt ended.
  • Oligonucleotide adapters can be ligated to the ends of the fragments.
  • the adapters can serve as primers for amplification and sequencing of the fragments.
  • the fragments can be attached to a surface and can be attached at a resolution such that the fragments are individually resolvable. Addition of one or more nucleotides can release a proton (H+ ), which signal detected and recorded in a sequencing instrument. The signal strength can be proportional to the number of nucleotides incorporated.
  • Illumina sequencing is based on the amplification of nucleic acids on a solid surface using fold-back PCR and anchored primers. Nucleic acids in a nucleic acid sample can be fragmented, and adapters can be added to the 5' and 3' ends of the fragments. Target nucleic acid fragments that are attached to the surface of flow cell channels can be extended and bridge amplified. The fragments can become double stranded, and the double stranded molecules are denatured.
  • SMRT real-time
  • each of the four DNA bases can be attached to one of four different fluorescent dyes. These dyes can be phospholinked.
  • a single DNA polymerase can be immobilized with a single molecule of template single stranded DNA at the bottom of a zero-mode waveguide (ZMW).
  • ZMW can be a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that rapidly diffuse in an out of the ZMW (in microseconds).
  • the fluorescent label can be excited and can produce a fluorescent signal, and the fluorescent tag can be cleaved off. Detection of the corresponding fluorescence of the dye can indicate which base was incorporated.
  • a nanopore can be a small hole, of the order of 1 nanometer in diameter.
  • Immersion of a nanopore in a conducting fluid and application of a potential across it can result in a slight electrical current due to conduction of ions through the nanopore.
  • the amount of current which flows can be sensitive to the size of the nanopore.
  • each nucleotide on the target nucleic acid can obstruct the nanopore to a different degree.
  • the change in the current passing through the nanopore as the target nucleic acid passes through the nanopore can represent a reading of the DNA sequence.
  • a sequencing technique that can be used in the methods of the provided invention can involve using a chemical-sensitive field effect transistor (chemFET) array to sequence a target nucleic acid.
  • chemFET chemical-sensitive field effect transistor
  • target nucleic acids can be placed into reaction chambers, and the template molecules can be hybridized to a sequencing primer bound to a polymerase.
  • Incorporation of one or more triphosphates into a new nucleic acid strand at the 3' end of the sequencing primer can be detected by a change in current by a chemFET.
  • An array can have multiple chemFET sensors.
  • single nucleic acids can be attached to beads, and the nucleic acids can be amplified on the bead, and the individual beads can be transferred to individual reaction chambers on a chemFET array, with each chamber having a chemFET sensor, and the nucleic acids can be sequenced.
  • Another example of a sequencing technique that can be used in the methods of the provided invention can involve using an electron microscope.
  • individual target nucleic acids can be labeled using metallic labels that can be distinguishable using an electron microscope.
  • These nucleic acids can be stretched on a flat surface and imaged using an electron microscope to measure sequences.
  • the obtained sequence reads can be split according to their bar code, i.e., demultiplexed, and reads originating from individual wells can be saved into separate files. Fragments amplified within each partitioned portion can be reconstructed using a de-novo assembly or by aligning to known reference sequence if such sequence exists.
  • Methods of the present disclosure may take advantage of pair-end reads and sequencing quality scores that represent base calling confidence to reconstruct full length fragments.
  • short reads may be stitched together bioinformatically, e.g., by finding overlaps and extending them.
  • Pathogen detection analysis can be performed after the nucleic acid sample is sequenced. Pathogen detection analysis can be performed with a computation program (e.g., Scalable Nucleotide Alignment Project (SNAP), Sequedex). Computation can align 100 million reads in about 2 minutes. A data analysis program can be customized. A data analysis program can use high-throughput, low resource methods to analyze pathogen phylogeny.
  • SNAP Scalable Nucleotide Alignment Project
  • Sequedex Sequedex
  • Computation can align 100 million reads in about 2 minutes.
  • a data analysis program can be customized.
  • a data analysis program can use high-throughput, low resource methods to analyze pathogen phylogeny.
  • a data analysis program can subtract the human background reads.
  • a data analysis program can have a specificity of greater than 70%, 80%, 85%, 90%, 95%, 97%, 99% specificity in mapping reads.
  • a data analysis program can have a sensitivity of greater than 70%, 80%, 85%, 90%, 95%, 97%, 99% sensitivity in mapping reads.
  • Pathogen detection analysis can be performed in less than 6 months, 3 months, 1 month, 3 weeks, 2 weeks, 1 week, 3 days, 2 days, lday 12 hours, 7 hours, 3 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute. Pathogen detection analysis can be performed in 2 minutes.
  • the Ultra-Rapid Pathogen Identification in Minutes can comprise obtaining raw next generation sequence read data (e.g., deep sequencing data), and performing a comparison to a viral and/or bacterial database using the Sequedex program, and then comparing the results to a human database using the SNAP program. Comparison to the human database can remove host genes that can contribute to background noise. The resulting genes can be compared to a bacterial database using the SNAP program. This method can identify genes of bacterial origin. To determine genes of viral origin, the results obtained after comparision to the human database can be compared to a viral database (also using SNAP).
  • a viral database also using SNAP
  • results obtained after comparison to the human database can be compared to nucleotides from all pathogenic databases.
  • the identified sequences can be assembled by de novo contig assembly and/or seed-based contig assembly methods. Host response analysis
  • Host response analysis can evaluate the changes in expression levels of genes associated with tick- borne diseases.
  • a data analysis program for determining host response changes e.g., Protein Analysis Through Evolutionary Relationships (PANTHER)
  • PANTHER Protein Analysis Through Evolutionary Relationships
  • a data analysis program can compare the gene expression changes in the levels of cytokines from an experimental sample and a control sample.
  • a nucleic acid sample can comprise nucleic acids from pathogens (e.g., bacteria, protozoa, fungi,
  • Host nucleic acids can be a contaminant that can lower the signal-to- noise ratio of the sequencing data from the pathogens, making it difficult to detect pathogenic sequences.
  • Host response analysis can group nucleic acids originating from the host and subtract them from the total nucleic acid sample, thus leaving the pathogenic nucleic acids for analysis.
  • the methods of the present disclosure can be computer-implemented, such that method steps (e.g., assaying, comparing, calculating, and the like) are be automated in whole or in part. Accordingly, the present disclosure provides methods, computer systems, devices and the like in connection with computer-implemented methods of facilitating a diagnosis of tick-borne diseae.
  • the method steps including obtaining information regarding detection of a pathogen
  • polynucleotide in a biological sample, generating a report, and the like can be completely or partially performed by a computer program product. Values obtained can be stored electronically, e.g., in a database, and can be subjected to an algorithm executed by a programmed computer.
  • the methods of the present disclosure can involve inputting information regarding
  • a pathogen polynucleotide in a biological sample into a computer programmed to execute an algorithm to perform the comparing and calculating step(s) described herein, and generate a report as described herein, e.g., by displaying or printing a report to an output device at a location local or remote to the computer.
  • the present invention thus provides a computer program product including a computer readable storage medium having a computer program stored on it.
  • the program can, when read by a computer, execute relevant calculations based on values obtained from analysis of one or more biological sample from an individual.
  • the computer program product has stored therein a computer program for performing the calculation(s).
  • a central computing environment generally includes: a) a central computing environment; b) an input device, operatively connected to the computing environment, to receive patient data, wherein the patient data can include, for example, biomarker level or other value obtained from an assay using a biological sample from the patient, as described above; c) an output device, connected to the computing environment, to provide information to a user (e.g., medical personnel); and d) an algorithm executed by the central computing environment (e.g., a processor), where the algorithm is executed based on the data received by the input device, and wherein the algorithm calculates a value, which value is indicative of the likelihood the subject is infected with a tick-borne pathogen as described herein,
  • FIG. 4 A generalized example of a computerized embodiment in which programs to facilitate execution of the methods of the present disclosure can be implemented is depicted in Figure 4, which illustrates a processing system 100 which generally comprises at least one processor 102, or processing unit or plurality of processors, memory 104, at least one input device 106 and at least one output device 108, coupled together via a bus or group of buses 110.
  • input device 106 and output device 108 can be the same device.
  • An interface 112 can also be provided for coupling the processing system 100 to one or more peripheral devices, for example interface 112 can be a PCI card or PC card.
  • At least one storage device 114 which houses at least one database 116 can also be provided.
  • the memory 104 can be any form of memory device, for example, volatile or non- volatile memory, solid state storage devices, magnetic devices, etc.
  • the processor 102 can comprise more than one distinct processing device, for example to handle different functions within the processing system 100.
  • Input device 106 receives input data 118 and can comprise, for example, a keyboard, a pointer device such as a pen-like device or a mouse, audio receiving device for voice controlled activation such as a microphone, data receiver or antenna such as a modem or wireless data adaptor, data acquisition card, etc.
  • Input data 118 can come from different sources, for example keyboard instructions in conjunction with data received via a network.
  • Output device 108 produces or generates output data 120 and can comprise, for example, a display device or monitor in which case output data 120 is visual, a printer in which case output data 120 is printed, a port for example a USB port, a peripheral component adaptor, a data transmitter or antenna such as a modem or wireless network adaptor, etc.
  • Output data 120 can be distinct and derived from different output devices, for example a visual display on a monitor in conjunction with data transmitted to a network.
  • a user can view data output, or an interpretation of the data output, on, for example, a monitor or using a printer.
  • the storage device 114 can be any form of data or information storage means, for example, volatile or non-volatile memory, solid state storage devices, magnetic devices, etc.
  • the processing system 100 is adapted to allow data or information to be stored in and/or
  • the interface 112 may allow wired and/or wireless communication between the processing unit 102 and peripheral components that may serve a specialized purpose.
  • the processor 102 can receive instructions as input data 118 via input device 106 and can display processed results or other output to a user by utilizing output device 108. More than one input device 106 and/or output device 108 can be provided.
  • the processing system 100 may be any suitable form of terminal, server, specialized hardware, or the like.
  • the processing system 100 may be a part of a networked communications system.
  • Processing system 100 can connect to a network, for example the Internet or a WAN.
  • Input data 118 and output data 120 can be communicated to other devices via the network.
  • the transfer of information and/or data over the network can be achieved using wired communications means or wireless communications means.
  • a server can facilitate the transfer of data between the network and one or more databases.
  • a server and one or more databases provide an example of an information source.
  • the logical connections depicted in Figure 4 may include a local area network (LAN) and a wide area network (WAN), but may also include other networks such as a personal area network (PAN).
  • LAN local area network
  • WAN wide area network
  • PAN personal area network
  • Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet.
  • the computing system environment 100 is connected to the LAN through a network interface or adapter.
  • the computing system environment typically includes a modem or other means for establishing communications over the WAN, such as the Internet.
  • the modem which may be internal or external, may be connected to a system bus via a user input interface, or via another appropriate mechanism.
  • program modules depicted relative to the computing system environment 100, or portions thereof may be stored in a remote memory storage device.
  • Figure 4 is intended to provide a brief, general description of an illustrative and/or suitable example of a computing environment in which embodiments of the methods disclosed herein may be implemented.
  • Figure 4 is an example of a suitable environment and is not intended to suggest any limitation as to the structure, scope of use, or functionality of an embodiment of the present invention.
  • a particular environment should not be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in an exemplary operating environment. For example, in certain instances, one or more elements of an environment may be deemed not necessary and omitted. In other instances, one or more other elements may be deemed necessary and added.
  • Embodiments may be implemented with numerous other general-purpose or special-purpose computing devices and computing system environments or configurations.
  • Examples of well-known computing systems, environments, and configurations that may be suitable for use with an embodiment include, but are not limited to, personal computers, handheld or laptop devices, personal digital assistants, multiprocessor systems, microprocessor-based systems, programmable consumer electronics, network, minicomputers, server computers, web server computers, mainframe computers, and distributed computing environments that include any of the above systems or devices.
  • Embodiments may be described in a general context of computer-executable instructions, such as
  • program modules being executed by a computer.
  • program modules include routines, programs, objects, components, data structures, etc., that perform particular tasks or implement particular abstract data types.
  • An embodiment may also be practiced in a distributed computing environment where tasks are performed by remote processing devices that are linked through a communications network.
  • program modules may be located in both local and remote computer storage media including memory storage devices.
  • the present disclosure provides computer program products that, when executed on a programmable computer such as that described above with reference to Figure 4, can carry out the methods of the present disclosure.
  • the subject matter described herein may be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration.
  • These various implementations may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device (e.g. video camera, microphone, joystick, keyboard, and/or mouse), and at least one output device (e.g. display monitor, printer, etc.).
  • at least one input device e.g. video camera, microphone, joystick, keyboard, and/or mouse
  • at least one output device e.g. display monitor, printer, etc.
  • Computer programs include instructions for a programmable processor, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language.
  • machine -readable medium refers to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, etc.) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal.
  • processors such as a microprocessor, executing sequences of instructions stored in memory or other computer-readable medium including any type of ROM, RAM, cache memory, network memory, floppy disks, hard drive disk (HDD), solid-state devices (SSD), optical disk, CD- ROM, and magnetic-optical disk, EPROMs, EEPROMs, flash memory, or any other type of media suitable for storing instructions in electronic format.
  • processors such as a microprocessor, executing sequences of instructions stored in memory or other computer-readable medium including any type of ROM, RAM, cache memory, network memory, floppy disks, hard drive disk (HDD), solid-state devices (SSD), optical disk, CD- ROM, and magnetic-optical disk, EPROMs, EEPROMs, flash memory, or any other type of media suitable for storing instructions in electronic format.
  • the processor(s) may be, or may include, one or more programmable general-purpose or special-purpose microprocessors, digital signal processors (DSPs), programmable controllers, application specific integrated circuits (ASICs), programmable logic devices (PLDs), trusted platform modules (TPMs), or the like, or a combination of such devices.
  • DSPs digital signal processors
  • ASICs application specific integrated circuits
  • PLDs programmable logic devices
  • TPMs trusted platform modules
  • special- purpose hardware such as logic circuits or other hardwired circuitry may be used in combination with software instructions to implement the techniques described herein.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • EXAMPLE 1 EXAMPLE 1
  • a high-density 57,954-probe microarray was developed and called the TickChipTM (UCSF), for
  • TickChip Using probes to conserved and hypervariable regions of 16S and 18S ribosomal RNA TickChip is capable of detection of tick-borne bacterial, fungal, and parasitic pathogens. TickChip targets clinically relevant bacteria, fungi, and protozoa and includes not only tick-borne pathogens but other vector-borne pathogens and bloodborne pathogens of interest. A list of exemplary pathogens targeted by TickChip is provided in Table 1 :
  • Spirochetes all known Borrelia species and strains; Treponema spp., Leptospira spp
  • Protozoa Babesia (microti, duncani, others), Toxoplasma spp.
  • Viruses Tickbornc encephalitis virus, Powassan Virus, dengue virus, other bloodborne viral
  • Fungi Candida spp., Cryplococcus spp., Aspergillus Jiimigalus
  • whole blood samples (or serum/plasma, if whole blood is available) are extracted to RNA followed by RT-PCR amplification, fluorescent labeling of amplified cDNA, and hybridization to a TickChip microarray for simultaneous detection of all tick-borne pathogens.
  • Extracted nucleic acid was then subjected to 50 cycles of PCR amplification using a multiplexed set of primers targeting highly conserved 16S and 18S ribosomal sequences in bacteria, fungi, and parasites. Aminoallyl-labeled nucleotides were added to the PCR mixture to enable fluorescent dye incorporation of the amplified 16S/18S product.
  • TickChip microarrays were washed and scanned at 2 ⁇ resolution using an Agilent DNA Microarray Scanner. Samples were computationally analyzed using hierarchical clustering [3] and Z-score analysis, as previously described [2]. In cluster analysis, sets of probes are "clustered" together in an unbiased fashion. Heat map analysis of the clusters of samples and probes can reveal patterns in the data that can be easily visualized and interpreted (see Figure 3). In Z-score analysis, microarray intensities are ranked in order of statistical significance relative to a previously defined set of background arrays. In this case, background arrays were a set of TickChip arrays corresponding to blood from healthy negative control blood donors. A TickChip array was called positive if there were >5 probes specific for a given bacterial, viral, parasitic, or fungal genus / species.
  • the bacterial pathogens examined include Borrelia burgdorferi, Anaplasma phagocytophilum, and
  • B. microti is of especial concern as the most frequent transfusion-transmitted parasitic infection and currently viewed as one of the top threats to the safety of blood transfusion in the United States (Wormser, et al., 2011, Annals of Internal Medicine, 155(8):509-519).
  • Testing of the BloodChip with analysis of B. burgdorferi, B. microti, and A. phagocytophilum serially diluted in negative whole blood matrices at 10 5 -10° copies per mL is complete ( Figure 3).
  • PCR for 16S/18S rRNA followed by BloodChip microarray analysis yielded a sensitivity of 1 bacterial genome per mL ( Figure 3).
  • Figure 3A depicts a heat map showing a selected cluster of 42 Babesia 18S probes on TickChip vl .0 microarrays corresponding to serial spiked-in dilutions of Babesia microti or Borrelia burgdorferi. The color saturation indicates the normalized magnitude of hybridization intensity on the TickChip.
  • the signature from multiple Babesia 18S probes is highly specific for Babesia microti at dilutions down to 10° genomes/mL (i.e., 1 genome/ml, 1 organism per ml).
  • Figure 3B illustrates the measured sensitivity of the TickChip for pathogens spiked into human whole blood and amplified by random amplification or 16S or 18S rRNA PCR
  • the initial candidate probe set was selected. Theoretical coverage of the probes was computationally assessed and optimized for detection and species discrimination of each of the target pathogens (e.g. discriminating B. burgdorferi from B. miyamotoi). The final design included -60,000 70mer probes. Experiments were performed targeting the V3-V4 hypervariable region of the 16S rRNA gene produced which produced the strongest hybridization signature for B. burgdorferi. This also enabled detection of other tick-borne agents such as Rickettsia rickettsia and Francisella tularensis. Initial studies on B. burgdorferi and B. microti show sensitivity of this approach down to 1 copy per niL of sample ( Figure 21 and Figure 23). Sensitivity down to 1 copy per niL of sample was also achieved for Anaplasma phagocytophilum and dengue virus (Table 2).
  • Samples were as follows: Borrelia burgdorferi (spiked in human blood), Babesia microti (spiked in hamster blood), Anaplasma phagocytophilum (serial culture dilutions), Dengue virus (flavivirus) (high- titer viral infections spiked as dilutions in negative human blood matrix), Hazara virus (bunyavirus) (cultures spiked in human blood), Borrelia miyamotoi (cultures spiked in human blood).
  • Custom TickChip microarrays were synthesized by Agilent Technologies and microarrays processed for scanning using a 3 ⁇ Agilent DNA microarray scanner.
  • the amplified cDNA was labeled using Cy3 fluorescent dye and hybridized to the microarray overnight.
  • analysis for pathogen identification was performed using hierarchical cluster analysis and Z-score oligonucleotide analysis.
  • Figures 21-23 depict TickChip detection of Borrelia burgdorferi and Babesia microti in spiked and clinical samples at titers of 1-10 copies per mL.
  • Figure 21 depicts a cluster heat map of 52 Borrelia probes showing a microarray sensitivity of 10° for detection of B. burgodorferi by universal 16S rRNA amplification in whole blood samples spiked with known concentrations of the bacterium (10° - 10 4 ) or in serum from patients from Martha's Vineyard Hospital presenting with acute Lyme disease (MV1 and MV2).
  • the asterisks correspond to the probes plotted in Figure 22.
  • Figure 22 depicts the 7 probes comprising the positive signal in clinical samples MV1 and MV2.
  • the probe positions are plotted along the sequence of the B. burgdorferi 16S rRNA gene.
  • a Borrelia specific primer (Borrelia-specific-R) was added to the amplification mix to boost the detection sensitivity of the 16S universal primers (pan-16S-F/pan-16S-R).
  • Figure 23 depicts a cluster heat map of 80 Babesia probes showing a microarray sensitivity of 10° for detection of B. microti by universal 18S rRNA amplification in whole blood samples spiked with known concentrations of the parasite (10°— 10 4 ) or in previously quantified RBC lysates from blood donors screening positive for Babesia infection (C512, C514, and C542).
  • the TickChip microarray may be more sensitive than PCR at very low titers (-10°) as a positive signal in a PCR- negative sample (CS512) is observed (white oval).
  • a TickChip for increased discrimination between clinically significant species of Borrelia such as Borrelia burgodorferi and Borrelia miyamotoi
  • flaB gene based detection probes were added.
  • 272 70mer flaB detection probes were designed based on multiple sequence alignment of the flaB from the sequences of all Borrelia species available in GenBank ( Figures 24A-H).
  • a flaB RT-PCR which can be included as a separate reaction, multiplexed with the 16S rRNA assay, or performed serially, this allows species discrimination of Borrelia.
  • Staphylococcus epidermidis and Lactobacillus have been cultured for evaluating probe specificity.
  • 30 tick extracts that are positive for B. miyamotoi and B. bissetti have also been prepared.
  • Cultures of B. andersonii, B. miyamotoi, and the tickborne viruses Langat (flavivirus) and Hazara (bunyavirus) are also available.
  • Clinical samples also include 200 whole blood samples collected prior to doxycycline treatment from patients with acute or early Lyme confirmed by IFA and 80 samples of sera from patients with acute Lyme disease.
  • Microarrays are validated using the following pathogens as positive controls: Borrelia burgdorferi, Borrelia miyamotoi , Borrelia andersonii, Babesia microti, Colorado tick fever and dengue viruses (tick- and mosquito-borne viral pathogens, respectively), and Anaplasma phagocytophilum.
  • pathogens include Borrelia burgdorferi, Borrelia miyamotoi , Borrelia andersonii, Babesia microti, Colorado tick fever and dengue viruses (tick- and mosquito-borne viral pathogens, respectively), and Anaplasma phagocytophilum.
  • multiple types of Borrelia are included to test flaB based probes and subtyping of Borrelia at the species level.
  • Analytical sensitivity is assessed by probit analysis with a goal for limit of detection of 1 - 10 copies per niL. To evaluate reproducibility, 5 replicates near the limit of dection are performed.
  • TickChip To determine analytical specificity, positive samples not specifically targeted by the TickChip, including clinically relevant bacteria and fungi (Staphylococcus, Lactobacillus, Histoplasma, and Pneumocystis), are hybridized. The accuracy of the TickChip is evaluated relative to routine PCR, antibody, and immunofluorescent assays.
  • TickChip analytical sensitivity is performed using 4 common prototype tick-borne pathogens of clinical significance: B. burgdorferi, Babesia microti, Anaplasma phagocytophilum, and vector-borne dengue virus (mosquito) and Langat viruses (tick), with a goal sensitivity of 1-10 genome copies per mL of sample.
  • the lower limit of detection is first established, followed by evaluation of reproducibility near limits of detection, accuracy versus standard clinical testing using samples from available clinical cohorts of tick-borne disease, and specificity using a panel of negative control pathogens.
  • a user- friendly multiplexed protocol with the goals of rapid turnaround time (goal 12-24 hours) and ease of handling (e.g. sample handling and processing with robotic instruments) with no loss of sensitivity is developed through the optimization (to maintain sensitivity, accuracy, reproducibility, and specificity at targeted levels) of primer concentrations, ratios, and cycling conditions.
  • Internal and external spiked controls as included in the assay.
  • the protocol is designated a Standard Operating Protocol (SOP) and final protocol testing is performed in a CLIA-certified laboratory (e.g., the UCSF Clinical Microbiology Laboratory) to evaluate use of the fully validated TickChip assay for patient care.
  • SOP Standard Operating Protocol
  • CLIA-certified laboratory e.g., the UCSF Clinical Microbiology Laboratory
  • the TickChip microarray assay is directly compared, using clinical samples harboring known tick-borne / bloodborne pathogens, with conventional FDA-approved (e.g. 2-tier serology) and CLIA- validated (e.g. PCR) testing for these agents.
  • FDA-approved e.g. 2-tier serology
  • CLIA- validated e.g. PCR
  • Deep sequencing libraries for pathogen detection were prepared from clinical samples from patients with clinical acute Lyme disease as previously described [1,4]. Briefly, nucleic acid samples were randomly amplified from extracted material or 16S/18S PCR amplicons digested with fragmentase. DNA libraries for next-generation sequencing (NGS) were prepared using the Nextera kit (Illumina) and samples were deep sequenced on an Illumina MiSeq instrument. Samples were analyzed using an automated computational pipeline developed for pathogens by computational subtraction of host DNA and alignment to pathogen-specific databases (see below). This ultra-rapid pipeline uses computational subtraction to remove human host sequences and subsequent alignment to pathogen-specific databases at rates that far exceed what is available in the scientific literature. Using the pipeline, we have been able to identify Borrelia sequences in NGS data corresponding to patients with acute Lyme disease in underlO minutes.
  • Transcriptome analysis has revealed a candidate set of -20 diagnostic host response markers that are useful in discriminating between patients with acute Lyme disease, resolved infection, or PTLDS, including some previously known elements (e.g. TNF-a, IFITM2, SOCS) as well as novel, previously undescribed response genes.
  • SURPI is an already functional, cloud-compatible pipeline (Naccache, et al., manuscript in preparation; abstract presented at the 2013 ASM Biodefense and Emerging Diseases Meeting - http://www.asmbiodefense.org/index.php/final-program-pdf) (example of its use in Figures 17A-B) for ultra-rapid pathogen identification in NGS data.
  • SURPI uses the Sequedex algorithm [7] for initial screening of unprocessed reads by signature peptide matching, followed by SNAP [8], a fast nucleotide alignment tool, for computational subtraction of human host background sequences followed by in- depth pathogen identification.
  • FIG. 18 depicts a sample workflow for an NGS-based diagnostic assay.
  • HSV-1 herpes simplex virus 1
  • CSF cerebrospinal fluid
  • Sendout testing of CSF was positive for HSV-1 in 24 hr.
  • NGS with a similar turnaround time produced 0.18% (8,307) HSV-1 reads with coverage of 60%> of the 152 kB viral genome.
  • Borrelia burgdorferi sequences were detected in ⁇ 24 hours by NGS assay in a patient with unexplained fever and rash.

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Abstract

L'invention concerne des méthodes permettant de déterminer les identités génétiques de maladies transmises par les tiques, consistant à immobiliser une ou plusieurs molécules de polynucléotides au niveau de plusieurs positions sur un substrat, la molécule ou les molécules de polynucléotides donnant des informations génomiques provenant d'un ou plusieurs micro-organismes induisant les maladies transmises par les tiques sélectionnés dans le groupe comprenant des bactéries, des protozoaires, des champignons et des virus; à mettre un échantillon biologique en contact avec la ou les molécules de polynucléotides; obtenir des informations d'hybridation provenant du substrat suite à la mise en contact de l'échantillon biologique avec la ou les molécules biologiques; et à mettre les informations d'hybridation en rapport avec des informations liées à la maladie transmise par les tiques, ce qui permet de détecter la maladie transmise par les tiques selon une sensibilité au moins égale à 90 % ou selon une spécificité au moins égale à 90 %.
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CN115976178A (zh) * 2022-11-23 2023-04-18 山东大学 基于纳米孔宏基因组测序的sftsv检测方法
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EP3596237A4 (fr) * 2017-03-17 2021-01-27 Second Genome, Inc. Exploitation de données d'étude de communauté microbienne fécale basée sur une séquence pour identifier un biomarqueur composite pour le cancer colorectal
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CN110551839A (zh) * 2019-08-21 2019-12-10 中国农业科学院兰州兽医研究所 一种邓肯氏巴贝斯虫鉴别检测试剂盒及检测方法
ES2934850A1 (es) * 2021-08-24 2023-02-27 Servicio Andaluz De Salud METODO in vitro PARA EL DIAGNOSTICO DE UNA INFECCION POR Borrelia spp.
CN114058716A (zh) * 2021-12-08 2022-02-18 河套学院 蜱媒病原体多样性的分析方法
WO2024015879A1 (fr) * 2022-07-12 2024-01-18 The Regents Of The University Of California Identification des stades précoces de la maladie de lyme fondée sur l'expression génique
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