WO2014197607A1 - Detection of tick-borne diseases - Google Patents

Detection of tick-borne diseases 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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Prior art keywords
tick
nucleic acid
microchip
borne disease
borrelia
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French (fr)
Inventor
Charles Chiu
Andrea SWEI
Deanna LEE
Samia NACCACHE
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University of California Berkeley
University of California, San Diego
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University of California Berkeley
University of California, San Diego
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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
    • 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

This disclosure provides for methods to determine genetic identities of tick-borne diseases, 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-bome 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%.

Description

DETECTION OF TICK-BORNE DISEASES
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under Grant number HL105704 awarded by the National Institutes of Health. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/831,556, filed on June 5, 2013, and of U.S. Provisional Patent Application Serial No. 61/831,564 filed on June 5, 2013, the disclosures of which are herein incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED
AS A TEXT FILE
[0003] A Sequence Listing is provided herewith as a text file, "UCSF-483WO SeqList_ST25.txt" created on
June 1, 2014 and having a size of 14,904 KB. The contents of the text file are incorporated by reference herein in their entirety.
INTRODUCTION
[0004] 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.
[0005] The current estimates for infection statistics are often underreported due to the fact that Lyme disease and other tick-borne diseases can present as other more common diseases such as chronic fatigue syndrome, lupus, or other autoimmune diseases, but incidence of Lyme disease and other tick-borne diseases is increasing worldwide.
SUMMARY
[0006] In some embodiments, 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%. In some embodiments, 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
polynucleotide molecules. In some embodiments, the method further comprises sequencing at least a portion of the biological sample. In some embodiments, 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. In some embodiments, said tick- borne disease is detected at a sensitivity of at least 90%> and a specificity of at least 90%>.
[0007] In some embodiments, 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. In some embodiments, the bacteria include Borrelia burgdorferi, Anaplasma
phagocytophilum/Ehrlichia, Rickettsia, Bartonella, Tularemia or Leptospira. In some embodiments, the protozoans include Babesia microti, Babesia duncani or Toxoplasma gondii. In some embodiments, the fungi include Candida spp or Cryptococcus spp. In some embodiments, the viruses include tick-borne encephalitis virus or dengue virus, or a virus from the Bunyaviridae family of virses. In some embodiments, 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. In some embodiments, the number of the one or more polynucleotide molecules is at least 60,000. In some embodiments, the length of each of the one or more polynucleotide molecules is greater than 70 bp. In some embodiments, the length of each of the one or more polynucleotide molecules is less than 70 bp. In some embodiments, the tick-borne disease is Lyme Disease. In some embodiments, the tick-borne disease-inducing microorganisms comprise bacteria, protozoans and fungi. In some embodiments, 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.
[0008] In some embodiments, 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. In some embodiments, the tick-borne disease is Lyme Disease.
[0009] Additional aspects and advantages of the present disclosure will become readily apparent to those
skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
[0010] 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. In some embodiments, the biological sample is a blood sample. In some embodiments, the method further comprises RNA extraction and DNA extraction from the biological sample. In some embodiments, the method further comprises RNA extraction from the peripheral blood mononuclear cells (PBMC) found in the biological sample. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
[0011] In some embodiments, 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. In some embodiments, the method further comprises establishing a transcriptome of the biological sample. In some embodiments, 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.
[0012] In some embodiments, 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. In some embodiments, the detecting the tick-borne disease is at a sensitivity of at least 90% and at a specificity of at least 90%>.
[0013] 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%>.
[0014] The disclosure provides for a method of detecting a tick-borne disease 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 at a sensitivity of at least 90%> or at a specificity of at least 90%.
[0015] Additional aspects and advantages of the present disclosure will become readily apparent to those
skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
INCORPORATION BY REFERENCE
[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0018] FIGURE 1 depicts a method for determining expression levels of tick-borne disease genes in a host.
[0019] FIGURE 2 demonstrates an example of output from an analysis using the TickChip. Calculated Z
scores are included in the output of this example.
[0020] 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. As can be seen, 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).
[0021] 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
[0022] FIGURE4 provides 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.
[0023] FIGURE 5 depicts a method for determining expression levels of tick-borne disease genes in a host.
[0024] FIGURE 6 depicts a deep-sequencing bioinformatics pipeline for pathogen detection.
[0025] FIGURE 7 depicts the results from comparing the speed and sensitivity of various bioinformatics tools to process deep sequencing data.
[0026] FIGURE 8 depicts the analysis of sequences from four different patients, all with acute Lyme disease.
[0027] FIGURE 9 illustrates that computational challenge various based on the clinical sample type.
[0028] FIGURE 10 depicts the URPI pipeline for analyzing deep sequencing data.
[0029] FIGURES 11A-C depict various different pipelines for analyzing deep sequencing data
[0030] FIGURE 12 depicts the results from comparing the speed of the Sequedex method to other
bioinformatics tools to process deep sequencing data.
[0031] FIGURE 13 depicts the results from comparing the speed of various bioinformatics tools to process deep sequencing data.
[0032] FIGURE 14 depicts a comparison of Receiver Operating Characteristic (ROC) curves for various methods of mapping deep sequencing data.
[0033] FIGURE 15 illustrates that SOCS3 expression (a host gene) is increased in patients with Lyme disease relative to control patients.
[0034] 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).
[0035] FIGURES 17A-B depict the use of a Nanostring nCounter Multiplexed Analysis System for a deep- sequencing-based diagnostic assay (without amplification).
[0036] FIGURE 18 depicts a sample workflow for a deep-sequencing-based diagnostic assay.
[0037] FIGURE 19 depicts a diagram of a system (e.g., a computer system) for analyzing deep-sequencing data.
[0038] FIGURE 20 depicts an overview of one embodiment of the TickChip assay.
[0039] 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.
[0040] FIGURE 22 depicts the positions of particular probes along the B. burgdorferi 16S rRNA gene. [0041] 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.
[0042] FIGURES 24A-H provide 272 probe sequences based on flaB gene sequences from Borrelia species
(from top to bottom SEQ IDNOs: 58,002-58,273).
[0043] 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).
DETAILED DESCRIPTION
[0044] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. It shall be understood that different aspects of the invention can be appreciated individually, collectively, or in combination with each other.
[0045] As used herein, "microchip" generally refers to a high -density array of nucleic acid probes
immobilized in predetermined regions of a substrate. A microchip can also refer to a "microarray."
[0046] As used herein, "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. Such derivatives can include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. 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 (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS). 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. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, 111.; Fluorescein- 15-dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5- dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5- dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be, e.g., biotin-dNTP. Some non-limiting examples of 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."
[0047] The term "probe," as used herein, refers to a nucleic acid that is complementary to a nucleotide
sequence of interest. In certain cases, detection of a target analyte requires hybridization of a probe to a target. In certain embodiments, 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. In certain embodiments, a probe may be present on a surface of a planar support, e.g., in the form of an array.
[0048] As used herein, "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".
[0049] The terms "determining", "measuring", "evaluating", "assessing", "analyzing", and "assaying" are used interchangeably herein to refer to any form of measurement, and include 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.
General Overview
[0050] 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. As an alternative, hybridization may be detected by electrochemical or electrostatic methods.
[0051] 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-borne diseases
[0052] Tick-bome diseases can comprise a plurality of infection stages. Tick-bome diseases can comprise an acute infection stage. In an acute infection stage, a tick-bome disease (e.g., Lyme disease, Southern Tick- Associated Rash Illness) can exhibit symptoms of fever, achiness, and/or fatigue. In an acute infection stage, 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.
[0053] 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
califomiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia latyschewii, Borrelia lonestari , Borrelia lusitaniae, Borrelia mazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parked, Borrelia persica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, Borrelia vincentii). In some instances the tick-bome disease can originate from Borrelia burgdorferi.
[0054] Bacteria species can include species from the genus Anaplasma/Ehrlichia (e.g., Anaplasma
phagocytophilum, Ehrlichia phagocytophila, Anaplasma bovis, Anaplasma platys, Anaplasma marginale, Ehrlichia ewingii, Ehrlichia chaffeensis, Ehrlichia canis, Neorickettsia sennetsu). In some instances the tick-borne disease can originate from Anaplasma phagocytophilum/Erlichia.
[0055] Bacteria species can include species from the genus Rickettsia (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 parkeri, Rickettsia peacockii, Rickettsia prowazekii, Rickettsia hipicephaliRickettsia rickettsii, Rickettsia sibirica, Rickettsia slovaca, Rickettsia tamurae, Rickettsia typhi).
[0056] Bacteria species can include species from the genus Bartonella (e.g., Bartonella alsatica, Bartonella australis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis (also called weissii), 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 queenslandensis, Bartonella quintana, Bartonella rattaustraliani, Bartonella rattimassiliensis, Bartonella rochalimae, Bartonella schoenbuchensis, Bartonella silvatica, Bartonella silvicola, Bartonella talpae, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii spp. arupensis, Bartonella vinsonii spp. berkhoffii, Bartonella vinsonii spp. vinsonii, Bartonella phoceensis, Bartonella washoensis, Candidatus Bartonella antechini, Candidatus Bartonella bandicootii, Candidatus Bartonella breitschwerdtii, Candidatus Bartonella durdenii, Candidatus Bartonella eldjazairii, Candidatus Bartonella may otimonensis, Candidatus Bartonella melophagi, Candidatus Bartonella merieuxii, Candidatus Bartonella monaxi, Candidatus Bartonella rudakovii, Candidatus Bartonella tamiae, Candidatus Bartonella thailandensis, Candidatus Bartonella volans, Candidatus Bartonella woyliei).
[0057] Bacteria species can include species that are known to cause Tuleremia (e.g., Francisella tularensis).
[0058] Bacteria species can include species from the genus Leptospira (e.g, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira alexanderi, Leptospira weilii, 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 genomospecies 5).
[0059] 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,
Treponema pectinovorum, Treponema pedis, Treponema pertenue, Treponema porcinum, Treponema primitia, Treponema putidum, Treponema saccharophilum, Treponema socranskii, Treponema socranskii, Treponema socranskii, Treponema socranskii, Treponema stenostreptum, Treponema succinifaciens, Treponema zuelzerae).
Bacteria species can include species from the genus Mycoplasma (e.g., Mycoplasma adleri,
Mycoplasma agalactiae, Mycoplasma agassizii, Mycoplasma alkalescens, Mycoplasma alligatoris, Mycoplasma alvi, Mycoplasma amphoriforme, Mycoplasma anatis, Mycoplasma anseris, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma auris, Mycoplasma bovigenitalium, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasma buccale, Mycoplasma buteonis, Mycoplasma californicum, Mycoplasma canadense, Mycoplasma canis, Mycoplasma capricolum, Mycoplasma capricolum, Mycoplasma capricolum, Mycoplasma caviae, Mycoplasma cavipharyngis, Mycoplasma citelli, Mycoplasma cloacale, Mycoplasma coccoides, Mycoplasma collis, Mycoplasma columbinasale, Mycoplasma columbinum, Mycoplasma columborale, Mycoplasma conjunctivae, Mycoplasma corogypsi, Mycoplasma cottewii, Mycoplasma cricetuli, Mycoplasma crocodyli, Mycoplasma cynos, Mycoplasma dispar, Mycoplasma edwardii, Mycoplasma elephantis, Mycoplasma ellychniae, Mycoplasma equigenitalium, Mycoplasma equirhinis, Mycoplasma falconis, Mycoplasma fastidiosum, Mycoplasma faucium, Mycoplasma felifaucium, Mycoplasma feliminutum, Mycoplasma felis, Mycoplasma fermentans, Mycoplasma flocculare, Mycoplasma gallinaceum, Mycoplasma gallinarum, Mycoplasma gallisepticum, Mycoplasma gallopavonis, Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma glycophilum, Mycoplasma gypis, Mycoplasma haemocanis, Mycoplasma haemofelis, Mycoplasma haemomuris, Mycoplasma hominis, Mycoplasma hyopharyngis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma iguanae,
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 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 testudineum, Mycoplasma testudinis, Mycoplasma verecundum, Mycoplasma wenyonii, Mycoplasma yeatsii). [0061] Bacteria species can include species from the genus Chlamydia (e.g., Chlamydia muridarum,
Chlamydia pecorum, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia suis, Chlamydia trachomatis).
[0062] 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). In some instances, the tick-borne disease can originate from Babesia microti. In some instances, the tick-borne disease can originate from Babesia duncani.
[0063] Prozoans can include species from the genus Toxoplasma (e.g., Toxoplasma gondii). In some
instances, the tick-borne disease can originate from Toxoplasma gondii.
[0064] 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 oleophila, Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candida rugosa, Candida sake, Candida shehatea, Candida temnochilae, Candida tenuis, Candida theae, Candida tropicalis, Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candida subhashii, Candida viswanathii, Candida utilis).
[0065] 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).
[0066] 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
Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus (e.g., severe fever with thrombocytopenia syndrome (SFTS)), Tospovirus). 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.
[0067] Many genes can be related (e.g., affected, expression level altered) to tick-borne diseases. 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).
[0068] "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.
[0069] The IFIT2 polypeptide sequence is:
[0070] MSENNKNSLESSLRQLKCHFTWNLMEGENSLDDFEDKVFYRTEFQNREFKATMCNLLAYLKH LKGQNEAALECLRKAEELIQQEHADQAEIRSLVTWGNYAWVYYHMGRLSDVQIYVDKVKHV CEKFSSPYRIESPELDCEEGWTRLKCGGNQNERAKVCFEKALEKKPKNPEFTSGLAIASYRLDN WPPSQNAIDPLRQAIRLNPDNQYLKVLLALKLHKMREEGEEEGEGEKLVEEALEKAPGVTDVL RSAAKFYRRKDEPDKAIELLKKALEYIPNNAYLHCQIGCCYRAKVFQVMNLRENGMYGKRKL LELIGHAVAHLKKADEANDNLFRVCSILASLHALADQYEDAEYYFQKEFSKELTPVAKQLLHL RYGNFQLYQMKCEDKAIHHFIEGVKINQKSREKEKMKDKLQKIAKMRLSKNGADSEALHVLA FLQELNEKMQQADEDSERGLESGSLIPSASSWNGE (SEQ ID NO: 57,959)
[0071] The IFIT2 nucleic acid sequence is:
[0072] AGTTTCACTTTCCCTTTTGTAACGTCAGCTGAAGGGAAACAAACAAAAAGGAACCAGAGG CCACTTGTATATATAGGTCTCTTCAGCATTTATTGGTGGCAGAAGAGGAAGATTTCTGAAG AGTGCAGCTGCCTGAACCGAGCCCTGCCGAACAGCTGAGAATTGCACTGCAACCATGAGT GAGAACAATAAGAATTCCTTGGAGAGCAGCCTACGGCAACTAAAATGCCATTTCACCTGG AACTTGATGGAGGGAGAAAACTCCTTGGATGATTTTGAAGACAAAGTATTTTACCGGACT GAGTTTCAGAATCGTGAATTCAAAGCCACAATGTGCAACCTACTGGCCTATCTAAAGCACC TCAAAGGGCAAAACGAGGCAGCCCTGGAATGCTTACGTAAAGCTGAAGAGTTAATCCAGC AAGAGCATGCTGACCAGGCAGAAATCAGAAGTCTGGTCACCTGGGGAAACTATGCCTGGG TCTACTATCACATGGGCCGACTCTCAGACGTTCAGATTTATGTAGACAAGGTGAAACATGT CTGTGAGAAGTTTTCCAGTCCCTATAGAATTGAGAGTCCAGAGCTTGACTGTGAGGAAGG GTGGACACGGTTAAAGTGTGGAGGAAACCAAAATGAAAGAGCGAAGGTGTGCTTTGAGA AGGCTCTGGAAAAGAAGCCAAAGAACCCAGAATTCACCTCTGGACTGGCAATAGCAAGCT ACCGTCTGGACAACTGGCCACCATCTCAGAACGCCATTGACCCTCTGAGGCAAGCCATTCG GCTGAATCCTGACAACCAGTACCTTAAAGTCCTCCTGGCTCTGAAGCTTCATAAGATGCGT GAAGAAGGTGAAGAGGAAGGTGAAGGAGAGAAGTTAGTTGAAGAAGCCTTGGAGAAAGC CCCAGGTGTAACAGATGTTCTTCGCAGTGCAGCCAAGTTTTATCGAAGAAAAGATGAGCC AGACAAAGCGATTGAACTGCTTAAAAAGGCTTTAGAATACATACCAAACAATGCCTACCT
GCATTGCCAAATTGGGTGCTGCTATAGGGCAAAAGTCTTCCAAGTAATGAATCTAAGAGA
GAATGGAATGTATGGGAAAAGAAAGTTACTGGAACTAATAGGACACGCTGTGGCTCATCT
GAAGAAAGCTGATGAGGCCAATGATAATCTCTTCCGTGTCTGTTCCATTCTTGCCAGCCTC
CATGCTCTAGCAGATCAGTATGAAGACGCAGAGTATTACTTCCAAAAGGAATTCAGTAAA
GAGCTTACTCCTGTAGCGAAACAACTGCTCCATCTGCGGTATGGCAACTTTCAGCTGTACC
AAATGAAGTGTGAAGACAAGGCCATCCACCACTTTATAGAGGGTGTAAAAATAAACCAGA
AATCAAGGGAGAAAGAAAAGATGAAAGACAAACTGCAAAAAATTGCCAAAATGCGACTT
TCTAAAAATGGAGCAGATTCTGAGGCTTTGCATGTCTTGGCATTCCTTCAGGAGCTGAATG
AAAAAATGCAACAAGCAGATGAAGACTCTGAGAGGGGTTTGGAGTCTGGAAGCCTCATCC
CTTCAGCATCAAGCTGGAATGGGGAATGAAGAATAGAGATGTGGTGCCCACTAGGCTACT
GCTGAAAGGGAGCTGAAATTCCTCCACCAAGTTGGTATTCAAAATATGTAATGACTGGTAT
GGCAAAAGATTGGACTAAGACACTGGCCATACCACTGGACAGGGTTATGTTAACACCTGA
ATTGCTGGGTCTTGAGAGAGCCCAAGGAGTTCTGGGAGAGGGACCAGATTGGGGGGTAGG
TCCACGGGCTTGGTGATAGAATTATTTCTCGATTGACTTCTTGAGTGCAATTTGAACTGTAA
CATTTGCTTAGTCACCTTTAGTGGAGTAATCTACTGGGCTTGTTTCTATATTTATATAAAGC
AGCCAAATCCTTCATGTAATATTGAAGTCCATTTTTGCAATGTTGTTCCATACTTGGAGTCA
TTTTGCATCCCATAGAGGTTAGTCCTGCATAGCCAGTAATGTGCTAAGTTCATCCAAAAGC
TGGCGGACCAAAGTCTAAATAGGGCTCAGTATCCCCCATCGCTTATCTCTGCCTCCTTCCTC
CTCCTTCCCAGTCTATCATCAACCTTGAGTATTCTACACAATGTGAATTCAAGTGCCTGATT
AATTGAGGTGGCAACATAGTTTGAGACGAGGGCAGAGAACAGGAAGATACATAGCTAGA
AGCGACGGGTACAAAAAGCAATGTGTACAAGAAGACTTTCAGCAAGTATACAGAGAGTTC
ACCTCTACTCTGCCCTCCTCATAGTCATAATGTAGCAAGTAAAGAATGAGAATGGATTCTG
TACAATACACTAGAAACCAACATAATGTATTTCTTTAAAACCTGTGTGAAAAAATAAATGT
TCCACCAGTAGGGATAGGGGAAAAGTAACCAAAAGAGAGAAAGAGAAAGGAATGCTGGT
TTATCTTTGTAGATTGTAATCGAATGGAGAAATTTGCAGTATTTTAGCCACTATTAGGAATT
CGGTAGGAAAGCAACAAAACGTGGGAACCTGGTGACTAAGGGCCTGGTGCAAGGACTTG
GGAAATGTCATTGATAATAGATGGTGGGGTTTTCCCCCCTTTAGAAATGTTGGATATTAAG
TGATATAAACACTTCTTTTAACTCCGAAAATCTTCTGAGAAATCACAAAATTCACGGTATG
CTTGGAACGATTGAGATTTTCTAGGTAGATGCTGAATAGCCTAGACATCAAAGTTGGTGTG
AACCAAAATAGAGTCAGCTGACCCAGCATCAGCCACACTCTGGGTTGGAAAATGTTTGCC
TGTTGGAATTAATTTAAGCTTAAGTATATATCAACATTATTTTATTGTGCAATTAAAACAAT
TTTCTGTATTAAACATCTTGTTGCACGCATTTGAGGTCATCAGGGTGCAAAATTTGTATTCC TGAAAATGTCATATATTTTCATTAATAAATAACCTAAATATGATAAAACATAAAGCAGTGT TCTGGTTCATCTGGAATTTTGCTGTACTTTAAATCTTTCAGACTCAGCTACTGATAAATGAA ACGTTACACAGGTGTGAACCAAATCCAAATAACCTCGACTGGTCTACTATCATAATCACCT GAACAGAACAAAACTTTTTCCTCAGCTTTAAGAGTCCAGGGCTTCGGATAACAGCTGCCAT CTGCCACCTGCTACCATTGACCTACGTGAACACAGACATTCTGTCTCCACCTTGATGGTGG GTGGGCTGCTCCCCTTTTCTTTGTTAAATTTTGTGCTTTCATCACATTTTCTCTATTCTGACC TCTGTTATGAGAAATAAAAGTCACTGATTCCATTTTAAAAAAAAAAA (SEQ ID NO: 57,960)
[0073] "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.
[0074] The IFITM2 polypeptide sequence is:
[0075] MNHIVQTFSPWSGQPPNYEMLKEEQEVAMLGVPHNPAPPMSTVIHIRSETSVPDHVVWSLFN TLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGIFMTILLIIIPVLVV QAQR (SEQ ID NO: 57,961)
[0076] The IFITM2 nucleic acid sequence is:
[0077] GAGGAAACTGTTGAGAAAACGGAACTACTGGGGAAAGGGAGGGCTCACTGAGAACCATC CCGGTAACCCGATCACCGCTGGTCACCATGAACCACATTGTGCAAACCTTCTCTCCTGTCA ACAGCGGCCAGCCTCCCAACTACGAGATGCTCAAGGAGGAGCAGGAAGTGGCTATGCTGG GGGTGCCCCACAACCCTGCTCCCCCGATGTCCACCGTGATCCACATCCGCAGCGAGACCTC CGTGCCTGACCATGTGGTCTGGTCCCTGTTCAACACCCTCTTCATGAACACCTGCTGCCTGG GCTTCATAGCATTCGCGTACTCCGTGAAGTCTAGGGACAGGAAGATGGTTGGCGACGTGA CCGGGGCCCAGGCCTATGCCTCCACCGCCAAGTGCCTGAACATCTGGGCCCTGATTTTGGG CATCTTCATGACCATTCTGCTCATCATCATCCCAGTGTTGGTCGTCCAGGCCCAGCGATAG ATCAGGAGGCATCATTGAGGCCAGGAGCTCTGCCCGTGACCTGTATCCCACGTACTCTATC TTCCATTCCTCGCCCTGCCCCCAGAGGCCAGGAGCTCTGCCCTTGACCTGTATTCCACTTAC TCCACCTTCCATTCCTCGCCCTGTCCCCACAGCCGAGTCCTGCATCAGCCCTTTATCCTCAC ACGCTTTTCTACAATGGCATTCAATAAAGTGTATATGTTTCTGGTGCTGCTGTGACTTCAAA AAAAAA (SEQ ID NO: 57,962)
[0078] "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.
[0079] The SOCS 1 polypeptide sequence is:
[0080] MVAHNQ VAADNAVST AAEPRPvRPEP S S S S S S SP AAPARPRPCPAVPAP APGDTHFRTFRSHAD YRRITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRDSRQRNCFFALSVKMASGPTSI RVHFQAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRVRPLQELCRQRIVATVGRE NLARIPLNPVLRDYLS SFPFQI (SEQ ID NO: 57,963)
[0081] The SOCS 1 nucleic acid sequence is:
[0082] GGCAGCTGCACGGCTCCTGGCCCCGGAGCATGCGCGAGAGCCGCCCCGGAGCGCCCCGGA GCCCCCCGCCGTCCCGCCCGCGGCGTCCCGCGCCCCGCCGCCAGCGCACCCCCGGACGCTA TGGCCCACCCCTCCGGCTGGCCCCTTCTGTAGGATGGTAGCACACAACCAGGTGGCAGCCG ACAATGCAGTCTCCACAGCAGCAGAGCCCCGACGGCGGCCAGAACCTTCCTCCTCTTCCTC CTCCTCGCCCGCGGCCCCCGCGCGCCCGCGGCCGTGCCCCGCGGTCCCGGCCCCGGCCCCC GGCGACACGCACTTCCGCACATTCCGTTCGCACGCCGATTACCGGCGCATCACGCGCGCCA GCGCGCTCCTGGACGCCTGCGGATTCTACTGGGGGCCCCTGAGCGTGCACGGGGCGCACG AGCGGCTGCGCGCCGAGCCCGTGGGCACCTTCCTGGTGCGCGACAGCCGCCAGCGGAACT GCTTTTTCGCCCTTAGCGTGAAGATGGCCTCGGGACCCACGAGCATCCGCGTGCACTTTCA GGCCGGCCGCTTTCACCTGGATGGCAGCCGCGAGAGCTTCGACTGCCTCTTCGAGCTGCTG GAGCACTACGTGGCGGCGCCGCGCCGCATGCTGGGGGCCCCGCTGCGCCAGCGCCGCGTG CGGCCGCTGCAGGAGCTGTGCCGCCAGCGCATCGTGGCCACCGTGGGCCGCGAGAACCTG GCTCGCATCCCCCTCAACCCCGTCCTCCGCGACTACCTGAGCTCCTTCCCCTTCCAGATTTG ACCGGCAGCGCCCGCCGTGCACGCAGCATTAACTGGGATGCCGTGTTATTTTGTTATTACT TGCCTGGAACCATGTGGGTACCCTCCCCGGCCTGGGTTGGAGGGAGCGGATGGGTGTAGG GGCGAGGCGCCTCCCGCCCTCGGCTGGAGACGAGGCCGCAGACCCCTTCTCACCTCTTGAG GGGGTCCTCCCCCTCCTGGTGCTCCCTCTGGGTCCCCCTGGTTGTTGTAGCAGCTTAACTGT ATCTGGAGCCAGGACCTGAACTCGCACCTCCTACCTCTTCATGTTTACATATACCCAGTAT CTTTGCACAAACCAGGGGTTGGGGGAGGGTCTCTGGCTTTATTTTTCTGCTGTGCAGAATC CTATTTTATATTTTTTAAAGTCAGTTTAGGTAATAAACTTTATTATGAAAGTTTTTTTTTT
(SEQ ID NO: 57,964)
[0083] "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.
[0084] The SOCS3 polypeptide sequence is:
[0085] MVTHSKFPAAGMSRPLDTSLRLKTFSSKSEYQLWNAVRKLQESGFYWSAVTGGEANLLLSA EPAGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPVPRFDCVLKLVHHY MPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGGEKIPLVLSRPLSSNVATLQHLCR KTVNGHLDSYEKVTQLPGPIREFLDQYDAPL (SEQ ID NO: 57,965)
[0086] The SOCS3 nucleic acid sequence is:
[0087] GGCTCCGACTTGGACTCCCTGCTCCGCTGCTGCCGCTTCGGCCCCGCACGCAGCCAGCCGC CAGCCGCCCGCCCGGCCCAGCTCCCGCCGCGGCCCCTTGCCGCGGTCCCTCTCCTGGTCCC CTCCCGGTTGGTCCGGGGGTGCGCAGGGGGCAGGGCGGGCGCCCAGGGGAAGCTCGAGG GACGCGCGCGCGAAGGCTCCTTTGTGGACTTCACGGCCGCCAACATCTGGGCGCAGCGCG GGCCACCGCTGGCCGTCTCGCCGCCGCGTCGCCTTGGGGACCCGAGGGGGCTCAGCCCCA AGGACGGAGACTTCGATTCGGGACCAGCCCCCCGGGATGCGGTAGCGGCCGCTGTGCGGA GGCCGCGAAGCAGCTGCAGCCGCCGCCGCGCAGATCCACGCTGGCTCCGTGCGCCATGGT CACCCACAGCAAGTTTCCCGCCGCCGGGATGAGCCGCCCCCTGGACACCAGCCTGCGCCTC AAGACCTTCAGCTCCAAGAGCGAGTACCAGCTGGTGGTGAACGCAGTGCGCAAGCTGCAG GAGAGCGGCTTCTACTGGAGCGCAGTGACCGGCGGCGAGGCGAACCTGCTGCTCAGTGCC GAGCCCGCCGGCACCTTTCTGATCCGCGACAGCTCGGACCAGCGCCACTTCTTCACGCTCA GCGTCAAGACCCAGTCTGGGACCAAGAACCTGCGCATCCAGTGTGAGGGGGGCAGCTTCT CTCTGCAGAGCGATCCCCGGAGCACGCAGCCCGTGCCCCGCTTCGACTGCGTGCTCAAGCT GGTGCACCACTACATGCCGCCCCCTGGAGCCCCCTCCTTCCCCTCGCCACCTACTGAACCC TCCTCCGAGGTGCCCGAGCAGCCGTCTGCCCAGCCACTCCCTGGGAGTCCCCCCAGAAGA GCCTATTACATCTACTCCGGGGGCGAGAAGATCCCCCTGGTGTTGAGCCGGCCCCTCTCCT CCAACGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGGACTCCTA TGAGAAAGTCACCCAGCTGCCGGGGCCCATTCGGGAGTTCCTGGACCAGTACGATGCCCC GCTTTAAGGGGTAAAGGGCGCAAAGGGCATGGGTCGGGAGAGGGGACGCAGGCCCCTCT CCTCCGTGGCACATGGCACAAGCACAAGAAGCCAACCAGGAGAGAGTCCTGTAGCTCTGG GGGGAAAGAGGGCGGACAGGCCCCTCCCTCTGCCCTCTCCCTGCAGAATGTGGCAGGCGG ACCTGGAATGTGTTGGAGGGAAGGGGGAGTACCACCTGAGTCTCCAGCTTCTCCGGAGGA GCCAGCTGTCCTGGTGGGACGATAGCAACCACAAGTGGATTCTCCTTCAATTCCTCAGCTT CCCCTCTGCCTCCAAACAGGGGACACTTCGGGAATGCTGAACTAATGAGAACTGCCAGGG AATCTTCAAACTTTCCAACGGAACTTGTTTGCTCTTTGATTTGGTTTAAACCTGAGCTGGTT GTGGAGCCTGGGAAAGGTGGAAGAGAGAGAGGTCCTGAGGGCCCCAGGGCTGCGGGCTG GCGAAGGAAATGGTCACACCCCCCGCCCACCCCAGGCGAGGATCCTGGTGACATGCTCCT CTCCCTGGCTCCGGGGAGAAGGGCTTGGGGTGACCTGAAGGGAACCATCCTGGTACCCCA CATCCTCTCCTCCGGGACAGTCACCGAAAACACAGGTTCCAAAGTCTACCTGGTGCCTGAG AGCCCAGGGCCCTTCCTCCGTTTTAAGGGGGAAGCAACATTTGGAGGGGATGGATGGGCT
GGTCAGCTGGTCTCCTTTTCCTACTCATACTATACCTTCCTGTACCTGGGTGGATGGAGCGG
GAGGATGGAGGAGACGGGACATCTTTCACCTCAGGCTCCTGGTAGAGAAGACAGGGGATT
CTACTCTGTGCCTCCTGACTATGTCTGGCTAAGAGATTCGCCTTAAATGCTCCCTGTCCCAT
GGAGAGGGACCCAGCATAGGAAAGCCACATACTCAGCCTGGATGGGTGGAGAGGCTGAG
GGACTCACTGGAGGGCACCAAGCCAGCCCACAGCCAGGGAAGTGGGGAGGGGGGGCGGA
AACCCATGCCTCCCAGCTGAGCACTGGGAATGTCAGCCCAGTAAGTATTGGCCAGTCAGG
CGCCTCGTGGTCAGAGCAGAGCCACCAGGTCCCACTGCCCCGAGCCCTGCACAGCCCTCCC
TCCTGCCTGGGTGGGGGAGGCTGGAGGTCATTGGAGAGGCTGGACTGCTGCCACCCCGGG
TGCTCCCGCTCTGCCATAGCACTGATCAGTGACAATTTACAGGAATGTAGCAGCGATGGAA
CAAACACAAAGTATTCTGTGTCAGGTATTGGGCTGGACAGGGCAGTTGTGTGTTGGGGTG
ACTCTGTCTTTTATAAAGATTCCACCTCCAGTCCTCTCTCCTCCCCCCTACTCAGGCCCTTG AGGCTATTAGGAGATGCTTGAAGAACTCAACAAAATCCCAATCCAAGTCAAACTTTGCAC ATATTTATATTTATATTCAGAAAAGAAACATTTCAGTAATTTATAATAAAGAGCACTATTT TTTAATGAAAAAAAAAAAAAAAAA (SEQ ID NO: 57,966)
[0088] "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.
[0089] The STAT1 (isoform alpha) polypeptide sequence is:
[0090] MSQWYELQQLDSKFLEQVHQLYDDSFPMEIRQYLAQWLEKQDWEHAANDVSFATIRFHDLLS QLDDQYSRFSLENNFLLQHNIRKSKRNLQDNFQEDPIQMSMIIYSCLKEERKILENAQRFNQAQ SGNIQSTVMLDKQKELDSKVRNVKDKVMCIEHEIKSLEDLQDEYDFKCKTLQNREHETNGVA KSDQKQEQLLLKKMYLMLDNKRKEVVHKIIELLNVTELTQNALINDELVEWKRRQQSACIGG PPNACLDQLQNWFTIVAESLQQVRQQLKKLEELEQKYTYEHDPITKNKQVLWDRTFSLFQQLI QSSFVVERQPCMPTHPQRPLVLKTGVQFTVKLRLLVKLQELNYNLKVKVLFDKDVNERNTVK GFRKFNILGTHTKVMNMEESTNGSLAAEFRHLQLKEQKNAGTRTNEGPLIVTEELHSLSFETQL CQPGLVIDLETTSLPVWISNVSQLPSGWASILWYNMLVAEPRNLSFFLTPPCARWAQLSEVLS WQFSSVTKRGLNVDQLNMLGEKLLGPNASPDGLIPWTRFCKENINDKNFPFWLWIESILELIKK HLLPLWNDGCIMGFISKERERALLKDQQPGTFLLRFSESSREGAITFTWVERSQNGGEPDFHAV EPYTKKELSAVTFPDIIRNYKVMAAENIPENPLKYLYPNIDKDHAFGKYYSRPKEAPEPMELDG PKGTGYIKTELISVSEVHPSRLQTTDNLLPMSPEEFDEVSRIVGSVEFDSMMNTV (SEQ ID NO: 57,967)
[0091] The STAT1 (isoform beta) polypeptide sequence is:
[0092] MSQWYELQQLDSKFLEQVHQLYDDSFPMEIRQYLAQWLEKQDWEHAANDVSFATIRFHDLLS QLDDQYSRFSLENNFLLQHNIRKSKRNLQDNFQEDPIQMSMIIYSCLKEERKILENAQRFNQAQ SGNIQSTVMLDKQKELDSKVRNVKDKVMCIEHEIKSLEDLQDEYDFKCKTLQNREHETNGVA KSDQKQEQLLLKKMYLMLDNKRKEVVHKIIELLNVTELTQNALINDELVEWKRRQQSACIGG PPNACLDQLQNWFTIVAESLQQVRQQLKKLEELEQKYTYEHDPITKNKQVLWDRTFSLFQQLI QSSFVVERQPCMPTHPQRPLVLKTGVQFTVKLRLLVKLQELNYNLKVKVLFDKDVNERNTVK GFRKFNILGTHTKVMNMEESTNGSLAAEFRHLQLKEQKNAGTRTNEGPLIVTEELHSLSFETQL CQPGLVIDLETTSLPVWISNVSQLPSGWASILWYNMLVAEPRNLSFFLTPPCARWAQLSEVLS WQFSSVTKRGLNVDQLNMLGEKLLGPNASPDGLIPWTRFCKENINDKNFPFWLWIESILELIKK HLLPLWNDGCIMGFISKERERALLKDQQPGTFLLRFSESSREGAITFTWVERSQNGGEPDFHAV EPYTKKELSAVTFPDIIRNYKVMAAENIPENPLKYLYPNIDKDHAFGKYYSRPKEAPEPMELDG PKGTGYIKTELISVSEV (SEQ ID NO: 57,968)
[0093] The STAT1 (isoform alpha) nucleic acid sequence is:
[0094] GCTGAGCGCGGAGCCGCCCGGTGATTGGTGGGGGCGGAAGGGGGCCGGGCGCCAGCGCT GCCTTTTCTCCTGCCGGGTAGTTTCGCTTTCCTGCGCAGAGTCTGCGGAGGGGCTCGGCTG CACCGGGGGGATCGCGCCTGGCAGACCCCAGACCGAGCAGAGGCGACCCAGCGCGCTCG GGAGAGGCTGCACCGCCGCGCCCCCGCCTAGCCCTTCCGGATCCTGCGCGCAGAAAAGTT TCATTTGCTGTATGCCATCCTCGAGAGCTGTCTAGGTTAACGTTCGCACTCTGTGTATATAA CCTCGACAGTCTTGGCACCTAACGTGCTGTGCGTAGCTGCTCCTTTGGTTGAATCCCCAGG CCCTTGTTGGGGCACAAGGTGGCAGGATGTCTCAGTGGTACGAACTTCAGCAGCTTGACTC AAAATTCCTGGAGCAGGTTCACCAGCTTTATGATGACAGTTTTCCCATGGAAATCAGACAG TACCTGGCACAGTGGTTAGAAAAGCAAGACTGGGAGCACGCTGCCAATGATGTTTCATTT GCCACCATCCGTTTTCATGACCTCCTGTCACAGCTGGATGATCAATATAGTCGCTTTTCTTT GGAGAATAACTTCTTGCTACAGCATAACATAAGGAAAAGCAAGCGTAATCTTCAGGATAA TTTTCAGGAAGACCCAATCCAGATGTCTATGATCATTTACAGCTGTCTGAAGGAAGAAAGG AAAATTCTGGAAAACGCCCAGAGATTTAATCAGGCTCAGTCGGGGAATATTCAGAGCACA GTGATGTTAGACAAACAGAAAGAGCTTGACAGTAAAGTCAGAAATGTGAAGGACAAGGTT ATGTGTATAGAGCATGAAATCAAGAGCCTGGAAGATTTACAAGATGAATATGACTTCAAA TGCAAAACCTTGCAGAACAGAGAACACGAGACCAATGGTGTGGCAAAGAGTGATCAGAA ACAAGAACAGCTGTTACTCAAGAAGATGTATTTAATGCTTGACAATAAGAGAAAGGAAGT AGTTCACAAAATAATAGAGTTGCTGAATGTCACTGAACTTACCCAGAATGCCCTGATTAAT GATGAACTAGTGGAGTGGAAGCGGAGACAGCAGAGCGCCTGTATTGGGGGGCCGCCCAAT GCTTGCTTGGATCAGCTGCAGAACTGGTTCACTATAGTTGCGGAGAGTCTGCAGCAAGTTC GGCAGCAGCTTAAAAAGTTGGAGGAATTGGAACAGAAATACACCTACGAACATGACCCTA TCACAAAAAACAAACAAGTGTTATGGGACCGCACCTTCAGTCTTTTCCAGCAGCTCATTCA
GAGCTCGTTTGTGGTGGAAAGACAGCCCTGCATGCCAACGCACCCTCAGAGGCCGCTGGT
CTTGAAGACAGGGGTCCAGTTCACTGTGAAGTTGAGACTGTTGGTGAAATTGCAAGAGCT
GAATTATAATTTGAAAGTCAAAGTCTTATTTGATAAAGATGTGAATGAGAGAAATACAGT
AAAAGGATTTAGGAAGTTCAACATTTTGGGCACGCACACAAAAGTGATGAACATGGAGGA
GTCCACCAATGGCAGTCTGGCGGCTGAATTTCGGCACCTGCAATTGAAAGAACAGAAAAA
TGCTGGCACCAGAACGAATGAGGGTCCTCTCATCGTTACTGAAGAGCTTCACTCCCTTAGT
TTTGAAACCCAATTGTGCCAGCCTGGTTTGGTAATTGACCTCGAGACGACCTCTCTGCCCG
TTGTGGTGATCTCCAACGTCAGCCAGCTCCCGAGCGGTTGGGCCTCCATCCTTTGGTACAA
CATGCTGGTGGCGGAACCCAGGAATCTGTCCTTCTTCCTGACTCCACCATGTGCACGATGG
GCTCAGCTTTCAGAAGTGCTGAGTTGGCAGTTTTCTTCTGTCACCAAAAGAGGTCTCAATG
TGGACCAGCTGAACATGTTGGGAGAGAAGCTTCTTGGTCCTAACGCCAGCCCCGATGGTCT
CATTCCGTGGACGAGGTTTTGTAAGGAAAATATAAATGATAAAAATTTTCCCTTCTGGCTT
TGGATTGAAAGCATCCTAGAACTCATTAAAAAACACCTGCTCCCTCTCTGGAATGATGGGT
GCATCATGGGCTTCATCAGCAAGGAGCGAGAGCGTGCCCTGTTGAAGGACCAGCAGCCGG
GGACCTTCCTGCTGCGGTTCAGTGAGAGCTCCCGGGAAGGGGCCATCACATTCACATGGGT
GGAGCGGTCCCAGAACGGAGGCGAACCTGACTTCCATGCGGTTGAACCCTACACGAAGAA
AGAACTTTCTGCTGTTACTTTCCCTGACATCATTCGCAATTACAAAGTCATGGCTGCTGAG
AATATTCCTGAGAATCCCCTGAAGTATCTGTATCCAAATATTGACAAAGACCATGCCTTTG
GAAAGTATTACTCCAGGCCAAAGGAAGCACCAGAGCCAATGGAACTTGATGGCCCTAAAG
GAACTGGATATATCAAGACTGAGTTGATTTCTGTGTCTGAAGTTCACCCTTCTAGACTTCA
GACCACAGACAACCTGCTCCCCATGTCTCCTGAGGAGTTTGACGAGGTGTCTCGGATAGTG
TCTGGCGACAGTTTTCCTTCTCATCTGTGATTCCCTCCTGCTACTCTGTTCCTTCACATCCTG
TGTTTCTAGGGAAATGAAAGAAAGGCCAGCAAATTCGCTGCAACCTGTTGATAGCAAGTG
AATTTTTCTCTAACTCAGAAACATCAGTTACTCTGAAGGGCATCATGCATCTTACTGAAGG
TAAAATTGAAAGGCATTCTCTGAAGAGTGGGTTTCACAAGTGAAAAACATCCAGATACAC
CCAAAGTATCAGGACGAGAATGAGGGTCCTTTGGGAAAGGAGAAGTTAAGCAACATCTAG
CAAATGTTATGCATAAAGTCAGTGCCCAACTGTTATAGGTTGTTGGATAAATCAGTGGTTA
TTTAGGGAACTGCTTGACGTAGGAACGGTAAATTTCTGTGGGAGAATTCTTACATGTTTTC
TTTGCTTTAAGTGTAACTGGCAGTTTTCCATTGGTTTACCTGTGAAATAGTTCAAAGCCAAG
TTTATATACAATTATATCAGTCCTCTTTCAAAGGTAGCCATCATGGATCTGGTAGGGGGAA
AATGTGTATTTTATTACATCTTTCACATTGGCTATTTAAAGACAAAGACAAATTCTGTTTCT
TGAGAAGAGAATATTAGCTTTACTGTTTGTTATGGCTTAATGACACTAGCTAATATCAATA
GAAGGATGTACATTTCCAAATTCACAAGTTGTGTTTGATATCCAAAGCTGAATACATTCTG
CTTTCATCTTGGTCACATACAATTATTTTTACAGTTCTCCCAAGGGAGTTAGGCTATTCACA
ACCACTCATTCAAAAGTTGAAATTAACCATAGATGTAGATAAACTCAGAAATTTAATTCAT AAAATTGGGAAAGGAGTAGAAAAAGCAGTAACTGACAACTTGAATAATACACCAGAGAT
AATATGAGAATCAGATCATTTCAAAACTCATTTCCTATGTAACTGCATTGAGAACTGCATA
TGTTTCGCTGATATATGTGTTTTTCACATTTGCGAATGGTTCCATTCTCTCTCCTGTACTTTT
CCTTATCACTGACACAAAAAGTAGATTAAGAGATGGGTTTGACAAGGTTCTTCCCTTTTAC
TGCAAATGCTGTATTCTTCTTTGGTGGAGATAAAGATTTCTTGAGTTTTGTTTTAAAATTAA AGCTAAAGTATCTGTATTGCATTAAATATAATATGCACACAGTGCTTTCCGTGGCACTGCA TACAATCTGAGGCCTCCTCTCTCAGTTTTTATATAGATGGCGAGAACCTAAGTTTCAGTTG ATTTTACAATTGAAATGACTAAAAAACAAAGAAGACAACATTAAAACAATATTGTTTCTA
CTGCACTAGCCAAGAGACTTTACTTTTAAGAAGTATTAAAATTCTAAAATTCAAAAAAAAA AAAAAAAAA (SEQ ID NO: 57,969)
[0095] The STAT1 (isoform beta) nucleic acid sequence is:
[0096] GCTGAGCGCGGAGCCGCCCGGTGATTGGTGGGGGCGGAAGGGGGCCGGGCGCCAGCGCT GCCTTTTCTCCTGCCGGGTAGTTTCGCTTTCCTGCGCAGAGTCTGCGGAGGGGCTCGGCTG CACCGGGGGGATCGCGCCTGGCAGACCCCAGACCGAGCAGAGGCGACCCAGCGCGCTCG GGAGAGGCTGCACCGCCGCGCCCCCGCCTAGCCCTTCCGGATCCTGCGCGCAGAAAAGTT TCATTTGCTGTATGCCATCCTCGAGAGCTGTCTAGGTTAACGTTCGCACTCTGTGTATATAA CCTCGACAGTCTTGGCACCTAACGTGCTGTGCGTAGCTGCTCCTTTGGTTGAATCCCCAGG CCCTTGTTGGGGCACAAGGTGGCAGGATGTCTCAGTGGTACGAACTTCAGCAGCTTGACTC AAAATTCCTGGAGCAGGTTCACCAGCTTTATGATGACAGTTTTCCCATGGAAATCAGACAG TACCTGGCACAGTGGTTAGAAAAGCAAGACTGGGAGCACGCTGCCAATGATGTTTCATTT GCCACCATCCGTTTTCATGACCTCCTGTCACAGCTGGATGATCAATATAGTCGCTTTTCTTT GGAGAATAACTTCTTGCTACAGCATAACATAAGGAAAAGCAAGCGTAATCTTCAGGATAA TTTTCAGGAAGACCCAATCCAGATGTCTATGATCATTTACAGCTGTCTGAAGGAAGAAAGG AAAATTCTGGAAAACGCCCAGAGATTTAATCAGGCTCAGTCGGGGAATATTCAGAGCACA GTGATGTTAGACAAACAGAAAGAGCTTGACAGTAAAGTCAGAAATGTGAAGGACAAGGTT ATGTGTATAGAGCATGAAATCAAGAGCCTGGAAGATTTACAAGATGAATATGACTTCAAA TGCAAAACCTTGCAGAACAGAGAACACGAGACCAATGGTGTGGCAAAGAGTGATCAGAA ACAAGAACAGCTGTTACTCAAGAAGATGTATTTAATGCTTGACAATAAGAGAAAGGAAGT AGTTCACAAAATAATAGAGTTGCTGAATGTCACTGAACTTACCCAGAATGCCCTGATTAAT GATGAACTAGTGGAGTGGAAGCGGAGACAGCAGAGCGCCTGTATTGGGGGGCCGCCCAAT GCTTGCTTGGATCAGCTGCAGAACTGGTTCACTATAGTTGCGGAGAGTCTGCAGCAAGTTC GGCAGCAGCTTAAAAAGTTGGAGGAATTGGAACAGAAATACACCTACGAACATGACCCTA TCACAAAAAACAAACAAGTGTTATGGGACCGCACCTTCAGTCTTTTCCAGCAGCTCATTCA GAGCTCGTTTGTGGTGGAAAGACAGCCCTGCATGCCAACGCACCCTCAGAGGCCGCTGGT
CTTGAAGACAGGGGTCCAGTTCACTGTGAAGTTGAGACTGTTGGTGAAATTGCAAGAGCT
GAATTATAATTTGAAAGTCAAAGTCTTATTTGATAAAGATGTGAATGAGAGAAATACAGT
AAAAGGATTTAGGAAGTTCAACATTTTGGGCACGCACACAAAAGTGATGAACATGGAGGA
GTCCACCAATGGCAGTCTGGCGGCTGAATTTCGGCACCTGCAATTGAAAGAACAGAAAAA
TGCTGGCACCAGAACGAATGAGGGTCCTCTCATCGTTACTGAAGAGCTTCACTCCCTTAGT
TTTGAAACCCAATTGTGCCAGCCTGGTTTGGTAATTGACCTCGAGACGACCTCTCTGCCCG
TTGTGGTGATCTCCAACGTCAGCCAGCTCCCGAGCGGTTGGGCCTCCATCCTTTGGTACAA
CATGCTGGTGGCGGAACCCAGGAATCTGTCCTTCTTCCTGACTCCACCATGTGCACGATGG
GCTCAGCTTTCAGAAGTGCTGAGTTGGCAGTTTTCTTCTGTCACCAAAAGAGGTCTCAATG
TGGACCAGCTGAACATGTTGGGAGAGAAGCTTCTTGGTCCTAACGCCAGCCCCGATGGTCT
CATTCCGTGGACGAGGTTTTGTAAGGAAAATATAAATGATAAAAATTTTCCCTTCTGGCTT
TGGATTGAAAGCATCCTAGAACTCATTAAAAAACACCTGCTCCCTCTCTGGAATGATGGGT
GCATCATGGGCTTCATCAGCAAGGAGCGAGAGCGTGCCCTGTTGAAGGACCAGCAGCCGG
GGACCTTCCTGCTGCGGTTCAGTGAGAGCTCCCGGGAAGGGGCCATCACATTCACATGGGT
GGAGCGGTCCCAGAACGGAGGCGAACCTGACTTCCATGCGGTTGAACCCTACACGAAGAA
AGAACTTTCTGCTGTTACTTTCCCTGACATCATTCGCAATTACAAAGTCATGGCTGCTGAG
AATATTCCTGAGAATCCCCTGAAGTATCTGTATCCAAATATTGACAAAGACCATGCCTTTG
GAAAGTATTACTCCAGGCCAAAGGAAGCACCAGAGCCAATGGAACTTGATGGCCCTAAAG
GAACTGGATATATCAAGACTGAGTTGATTTCTGTGTCTGAAGTGTAAGTGAACACAGAAG
AGTGACATGTTTACAAACCTCAAGCCAGCCTTGCTCCTGGCTGGGGCCTGTTGAAGATGCT
TGTATTTTACTTTTCCATTGTAATTGCTATCGCCATCACAGCTGAACTTGTTGAGATCCCCG
TGTTACTGCCTATCAGCATTTTACTACTTTAAAAAAAAAAAAAAAGCCAAAAACCAAATTT
GTATTTAAGGTATATAAATTTTCCCAAAACTGATACCCTTTGAAAAAGTATAAATAAAATG
AGCAAAAGTTGAT (SEQ ID NO: 57,970)
[0097] "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.
[0098] The STAT2 (isoform 1) polypeptide sequence is:
[0099] MAQWEMLQNLDSPFQDQLHQLYSHSLLPVDIRQYLAVWIEDQNWQEAALGSDDSKATMLFF HFLDQLNYECGRCSQDPESLLLQHNLRKFCRDIQPFSQDPTQLAEMIFNLLLEEKRILIQAQRAQ LEQGEPVLETPVESQQHEIESRILDLRAMMEKLVKSISQLKDQQDVFCFRYKIQAKGKTPSLDP HQTKEQKILQETLNELDKRRKEVLDASKALLGRLTTLIELLLPKLEEWKAQQQKACIRAPIDHG LEQLETWFTAGAKLLFHLRQLLKELKGLSCLVSYQDDPLTKGVDLRNAQVTELLQRLLHRAF WETQPCMPQTPHRPLILKTGSKFTVRTRLLVRLQEGNESLTVEVSIDRNPPQLQGFRKFNILTS NQKTLTPEKGQSQGLIWDFGYLTLVEQRSGGSGKGSNKGPLGVTEELHIISFTVKYTYQGLKQ ELKTDTLPWIISNMNQLSIAWASVLWFNLLSPNLQNQQFFSNPPKAPWSLLGPALSWQFSSYV GRGLNSDQLSMLRNKLFGQNCRTEDPLLSWADFTKRESPPGKLPFWTWLDKILELVHDHLKD LWNDGRIMGFVSRSQERRLLKKTMSGTFLLRFSESSEGGITCSWVEHQDDDKVLIYSVQPYTK EVLQSLPLTEIIRHYQLLTEENIPENPLRFLYPRIPRDEAFGCYYQEKVNLQERRKYLKHRLIVVS NRQVDELQQPLELKPEPELESLELELGLVPEPELSLDLEPLLKAGLDLGPELESVLESTLEPVIEP TLCMVSQTVPEPDQGPVSQPVPEPDLPCDLRHLNTEPMEIFRNCVKIEEIMPNGDPLLAGQNTV DEVYVSRPSHFYTDGPLMPSDF (SEQ ID NO: 57,971)
[00100] The STAT2 (isoform 2) polypeptide sequence is:
[00101] MAQWEMLQNLDSPFQDQLHQLYSHSLLPVDIRQYLAVWIEDQNWQEAALGSDDSKATMLFF HFLDQLNYECGRCSQDPESLLLQHNLRKFCRDIQDPTQLAEMIFNLLLEEKRILIQAQRAQLEQ GEPVLETPVESQQHEIESRILDLRAMMEKLVKSISQLKDQQDVFCFRYKIQAKGKTPSLDPHQT KEQKILQETLNELDKRRKEVLDASKALLGRLTTLIELLLPKLEEWKAQQQKACIRAPIDHGLEQ LETWFTAGAKLLFHLRQLLKELKGLSCLVSYQDDPLTKGVDLRNAQVTELLQRLLHRAFVVE TQPCMPQTPHRPLILKTGSKFTVRTRLLVRLQEGNESLTVEVSIDRNPPQLQGFRKFNILTSNQK TLTPEKGQSQGLIWDFGYLTLVEQRSGGSGKGSNKGPLGVTEELHIISFTVKYTYQGLKQELKT DTLPWIISNMNQLSIAWASVLWFNLLSPNLQNQQFFSNPPKAPWSLLGPALSWQFSSYVGRGL NSDQLSMLRNKLFGQNCRTEDPLLSWADFTKRESPPGKLPFWTWLDKILELVHDHLKDLWND GRIMGFVSRSQERRLLKKTMSGTFLLRFSESSEGGITCSWVEHQDDDKVLIYSVQPYTKEVLQS LPLTEIIRHYQLLTEENIPENPLRFLYPRIPRDEAFGCYYQEKVNLQERRKYLKHRLIVVSNRQV DELQQPLELKPEPELESLELELGLVPEPELSLDLEPLLKAGLDLGPELESVLESTLEPVIEPTLCM VSQTVPEPDQGPVSQPVPEPDLPCDLRHLNTEPMEIFRNCVKIEEIMPNGDPLLAGQNTVDEVY VSRPSHFYTDGPLMPSDF (SEQ ID NO: 57,972)
[00102] The STAT2 (isoform 1) nucleic acid sequence is:
[00103] TGGCAGCCAGTGTCGGGGTGGCGGCTGGGAATGGGGGCCGCTCCGGACTTCCGCTGCCAA CTACAAGGGGGCGGGTCCGAGGGGGGTTAGCCGAAGTTGTAGGCGGGGCGCGAGGTTCTA GTACCCGAGCTCATACTAGGGACGGGAAGTCGCGACCAGAGCCATTGGAGGGCGCGGGG ACTGCAACCCTAATCAGAGCCCAAATGGCGCAGTGGGAAATGCTGCAGAATCTTGACAGC CCCTTTCAGGATCAGCTGCACCAGCTTTACTCGCACAGCCTCCTGCCTGTGGACATTCGAC AGTACTTGGCTGTCTGGATTGAAGACCAGAACTGGCAGGAAGCTGCACTTGGGAGTGATG ATTCCAAGGCTACCATGCTATTCTTCCACTTCTTGGATCAGCTGAACTATGAGTGTGGCCGT TGCAGCCAGGACCCAGAGTCCTTGTTGCTGCAGCACAATTTGCGGAAATTCTGCCGGGACA TTCAGCCCTTTTCCCAGGATCCTACCCAGTTGGCTGAGATGATCTTTAACCTCCTTCTGGAA GAAAAAAGAATTTTGATCCAGGCTCAGAGGGCCCAATTGGAACAAGGAGAGCCAGTTCTC GAAACACCTGTGGAGAGCCAGCAACATGAGATTGAATCCCGGATCCTGGATTTAAGGGCT
ATGATGGAGAAGCTGGTAAAATCCATCAGCCAACTGAAAGACCAGCAGGATGTCTTCTGC
TTCCGATATAAGATCCAGGCCAAAGGGAAGACACCCTCTCTGGACCCCCATCAGACCAAA
GAGCAGAAGATTCTGCAGGAAACTCTCAATGAACTGGACAAAAGGAGAAAGGAGGTGCT
GGATGCCTCCAAAGCACTGCTAGGCCGATTAACTACCCTAATCGAGCTACTGCTGCCAAAG
TTGGAGGAGTGGAAGGCCCAGCAGCAAAAAGCCTGCATCAGAGCTCCCATTGACCACGGG
TTGGAACAGCTGGAGACATGGTTCACAGCTGGAGCAAAGCTGTTGTTTCACCTGAGGCAG
CTGCTGAAGGAGCTGAAGGGACTGAGTTGCCTGGTTAGCTATCAGGATGACCCTCTGACC
AAAGGGGTGGACCTACGCAACGCCCAGGTCACAGAGTTGCTACAGCGTCTGCTCCACAGA
GCCTTTGTGGTAGAAACCCAGCCCTGCATGCCCCAAACTCCCCATCGACCCCTCATCCTCA
AGACTGGCAGCAAGTTCACCGTCCGAACAAGGCTGCTGGTGAGACTCCAGGAAGGCAATG
AGTCACTGACTGTGGAAGTCTCCATTGACAGGAATCCTCCTCAATTACAAGGCTTCCGGAA
GTTCAACATTCTGACTTCAAACCAGAAAACTTTGACCCCCGAGAAGGGGCAGAGTCAGGG
TTTGATTTGGGACTTTGGTTACCTGACTCTGGTGGAGCAACGTTCAGGTGGTTCAGGAAAG
GGCAGCAATAAGGGGCCACTAGGTGTGACAGAGGAACTGCACATCATCAGCTTCACGGTC
AAATATACCTACCAGGGTCTGAAGCAGGAGCTGAAAACGGACACCCTCCCTGTGGTGATT
ATTTCCAACATGAACCAGCTCTCAATTGCCTGGGCTTCAGTTCTCTGGTTCAATTTGCTCAG
CCCAAACCTTCAGAACCAGCAGTTCTTCTCCAACCCCCCCAAGGCCCCCTGGAGCTTGCTG
GGCCCTGCTCTCAGTTGGCAGTTCTCCTCCTATGTTGGCCGAGGCCTCAACTCAGACCAGC
TGAGCATGCTGAGAAACAAGCTGTTCGGGCAGAACTGTAGGACTGAGGATCCATTATTGT
CCTGGGCTGACTTCACTAAGCGAGAGAGCCCTCCTGGCAAGTTACCATTCTGGACATGGCT
GGACAAAATTCTGGAGTTGGTACATGACCACCTGAAGGATCTCTGGAATGATGGACGCAT
CATGGGCTTTGTGAGTCGGAGCCAGGAGCGCCGGCTGCTGAAGAAGACCATGTCTGGCAC
CTTTCTACTGCGCTTCAGTGAATCGTCAGAAGGGGGCATTACCTGCTCCTGGGTGGAGCAC
CAGGATGATGACAAGGTGCTCATCTACTCTGTGCAACCGTACACGAAGGAGGTGCTGCAG
TCACTCCCGCTGACTGAAATCATCCGCCATTACCAGTTGCTCACTGAGGAGAATATACCTG
AAAACCCACTGCGCTTCCTCTATCCCCGAATCCCCCGGGATGAAGCTTTTGGGTGCTACTA
CCAGGAGAAAGTTAATCTCCAGGAACGGAGGAAATACCTGAAACACAGGCTCATTGTGGT
CTCTAATAGACAGGTGGATGAACTGCAACAACCGCTGGAGCTTAAGCCAGAGCCAGAGCT
GGAGTCATTAGAGCTGGAACTAGGGCTGGTGCCAGAGCCAGAGCTCAGCCTGGACTTAGA
GCCACTGCTGAAGGCAGGGCTGGATCTGGGGCCAGAGCTAGAGTCTGTGCTGGAGTCCAC
TCTGGAGCCTGTGATAGAGCCCACACTATGCATGGTATCACAAACAGTGCCAGAGCCAGA
CCAAGGACCTGTATCACAGCCAGTGCCAGAGCCAGATTTGCCCTGTGATCTGAGACATTTG
AACACTGAGCCAATGGAAATCTTCAGAAACTGTGTAAAGATTGAAGAAATCATGCCGAAT
GGTGACCCACTGTTGGCTGGCCAGAACACCGTGGATGAGGTTTACGTCTCCCGCCCCAGCC
ACTTCTACACTGATGGACCCTTGATGCCTTCTGACTTCTAGGAACCACATTTCCTCTGTTCT
TTTCATATCTCTTGCCCTTCCTACTCCTCATAGCATGATATTGTTCTCCAAGGATGGGAATC AGGCATGTGTCCCTTCCAAGCTGTGTTAACTGTTCAAACTCAGGCCTGTGTGACTCCATTG
GGGTGAGAGGTGAAAGCATAACATGGGTACAGAGGGGACAACAATGAATCAGAACAGAT
GCTGAGCCATAGGTCTAAATAGGATCCTGGAGGCTGCCTGCTGTGCTGGGAGGTATAGGG
GTCCTGGGGGCAGGCCAGGGCAGTTGACAGGTACTTGGAGGGCTCAGGGCAGTGGCTTCT
TTCCAGTATGGAAGGATTTCAACATTTTAATAGTTGGTTAGGCTAAACTGGTGCATACTGG
CATTGGCCCTTGGTGGGGAGCACAGACACAGGATAGGACTCCATTTCTTTCTTCCATTCCT
TCATGTCTAGGATAACTTGCTTTCTTCTTTCCTTTACTCCTGGCTCAAGCCCTGAATTTCTTC
TTTTCCTGCAGGGGTTGAGAGCTTTCTGCCTTAGCCTACCATGTGAAACTCTACCCTGAAG
AAGCTGGCTGTACCTGTTCCTCCCCCATAAAATGATCCTGCCAATCTAATGTGAGTGTGAA GCTTTGCACACTAGTTTATGCTACCTAGTCTCCACTTTCTCAATGCTTAGGAGACAGATCAC
CTGTTGCCCAGGCTAGAGTGCAATGGTGCAATCACAGCTCACTGCAGCCTCAACCTCCTGG GTTCAAGCAATCCTCCTACCTCAGCCTCCTGGGTAGCTAGCACCATGGCATGCGCCACCAT GCCCTATTTTTTTTTTTTAAAGACAGGGTCTTGCTATATTGCCCAGGCTGGTCTTGAACTGG GCTCAAGTGATCCTCACGCCTTGGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACTG
GGAGTGCAATGGTGTGATCTCGGCTCACTGCAACCTCCGCCTTCCGGGTTCAAGTGACTCT CCTGCCTCAGCCTCCCCAGTAGCTGGGATTACAGATCTGCACCACCATGCCCAGCTAATTT
CAAGTGATCTGTCCACCTCGGCCTCCCAGAGTGCTGGGATTACAGGCGTGAGCCACTGTTC
ACCTGAAAGGAAGGTTTCTATTCGTTGGTTGTGGACCTGGACAAAGTCCAAGTCTGTGGAA CTTAAAACCTTGAAGGTCTGTCATAGGACTCTGGACAATCTCACACCTTAGCTATTCCCAG GGAACCCCAGGGGGCAACTGACATTGCTCCAAGATGTTCTCCTGATGTAGCTTGAGATATA AAGGAAAGGCCCTGCACAGGTGGCTGTTTCTTGTCTGTTATGTCAGAGGAACAGTCCTGTT CAGAAAGGGGCTCTTCTGAGCAGAAATGGCTAATAAACTTTGTGCTGATCTGGAAAAAAA AAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 57,973)
[00104] The STAT2 (isoform 2) nucleic acid sequence is:
[00105] TGGCAGCCAGTGTCGGGGTGGCGGCTGGGAATGGGGGCCGCTCCGGACTTCCGCTGCCAA CTACAAGGGGGCGGGTCCGAGGGGGGTTAGCCGAAGTTGTAGGCGGGGCGCGAGGTTCTA GTACCCGAGCTCATACTAGGGACGGGAAGTCGCGACCAGAGCCATTGGAGGGCGCGGGG ACTGCAACCCTAATCAGAGCCCAAATGGCGCAGTGGGAAATGCTGCAGAATCTTGACAGC CCCTTTCAGGATCAGCTGCACCAGCTTTACTCGCACAGCCTCCTGCCTGTGGACATTCGAC AGTACTTGGCTGTCTGGATTGAAGACCAGAACTGGCAGGAAGCTGCACTTGGGAGTGATG ATTCCAAGGCTACCATGCTATTCTTCCACTTCTTGGATCAGCTGAACTATGAGTGTGGCCGT TGCAGCCAGGACCCAGAGTCCTTGTTGCTGCAGCACAATTTGCGGAAATTCTGCCGGGACA
TTCAGGATCCTACCCAGTTGGCTGAGATGATCTTTAACCTCCTTCTGGAAGAAAAAAGAAT
TTTGATCCAGGCTCAGAGGGCCCAATTGGAACAAGGAGAGCCAGTTCTCGAAACACCTGT
GGAGAGCCAGCAACATGAGATTGAATCCCGGATCCTGGATTTAAGGGCTATGATGGAGAA
GCTGGTAAAATCCATCAGCCAACTGAAAGACCAGCAGGATGTCTTCTGCTTCCGATATAAG
ATCCAGGCCAAAGGGAAGACACCCTCTCTGGACCCCCATCAGACCAAAGAGCAGAAGATT
CTGCAGGAAACTCTCAATGAACTGGACAAAAGGAGAAAGGAGGTGCTGGATGCCTCCAAA
GCACTGCTAGGCCGATTAACTACCCTAATCGAGCTACTGCTGCCAAAGTTGGAGGAGTGG
AAGGCCCAGCAGCAAAAAGCCTGCATCAGAGCTCCCATTGACCACGGGTTGGAACAGCTG
GAGACATGGTTCACAGCTGGAGCAAAGCTGTTGTTTCACCTGAGGCAGCTGCTGAAGGAG
CTGAAGGGACTGAGTTGCCTGGTTAGCTATCAGGATGACCCTCTGACCAAAGGGGTGGAC
CTACGCAACGCCCAGGTCACAGAGTTGCTACAGCGTCTGCTCCACAGAGCCTTTGTGGTAG
AAACCCAGCCCTGCATGCCCCAAACTCCCCATCGACCCCTCATCCTCAAGACTGGCAGCAA
GTTCACCGTCCGAACAAGGCTGCTGGTGAGACTCCAGGAAGGCAATGAGTCACTGACTGT
GGAAGTCTCCATTGACAGGAATCCTCCTCAATTACAAGGCTTCCGGAAGTTCAACATTCTG
ACTTCAAACCAGAAAACTTTGACCCCCGAGAAGGGGCAGAGTCAGGGTTTGATTTGGGAC
TTTGGTTACCTGACTCTGGTGGAGCAACGTTCAGGTGGTTCAGGAAAGGGCAGCAATAAG
GGGCCACTAGGTGTGACAGAGGAACTGCACATCATCAGCTTCACGGTCAAATATACCTAC
CAGGGTCTGAAGCAGGAGCTGAAAACGGACACCCTCCCTGTGGTGATTATTTCCAACATG
AACCAGCTCTCAATTGCCTGGGCTTCAGTTCTCTGGTTCAATTTGCTCAGCCCAAACCTTCA
GAACCAGCAGTTCTTCTCCAACCCCCCCAAGGCCCCCTGGAGCTTGCTGGGCCCTGCTCTC
AGTTGGCAGTTCTCCTCCTATGTTGGCCGAGGCCTCAACTCAGACCAGCTGAGCATGCTGA
GAAACAAGCTGTTCGGGCAGAACTGTAGGACTGAGGATCCATTATTGTCCTGGGCTGACTT
CACTAAGCGAGAGAGCCCTCCTGGCAAGTTACCATTCTGGACATGGCTGGACAAAATTCT
GGAGTTGGTACATGACCACCTGAAGGATCTCTGGAATGATGGACGCATCATGGGCTTTGTG
AGTCGGAGCCAGGAGCGCCGGCTGCTGAAGAAGACCATGTCTGGCACCTTTCTACTGCGC
TTCAGTGAATCGTCAGAAGGGGGCATTACCTGCTCCTGGGTGGAGCACCAGGATGATGAC
AAGGTGCTCATCTACTCTGTGCAACCGTACACGAAGGAGGTGCTGCAGTCACTCCCGCTGA
CTGAAATCATCCGCCATTACCAGTTGCTCACTGAGGAGAATATACCTGAAAACCCACTGCG
CTTCCTCTATCCCCGAATCCCCCGGGATGAAGCTTTTGGGTGCTACTACCAGGAGAAAGTT
AATCTCCAGGAACGGAGGAAATACCTGAAACACAGGCTCATTGTGGTCTCTAATAGACAG
GTGGATGAACTGCAACAACCGCTGGAGCTTAAGCCAGAGCCAGAGCTGGAGTCATTAGAG
CTGGAACTAGGGCTGGTGCCAGAGCCAGAGCTCAGCCTGGACTTAGAGCCACTGCTGAAG
GCAGGGCTGGATCTGGGGCCAGAGCTAGAGTCTGTGCTGGAGTCCACTCTGGAGCCTGTG
ATAGAGCCCACACTATGCATGGTATCACAAACAGTGCCAGAGCCAGACCAAGGACCTGTA
TCACAGCCAGTGCCAGAGCCAGATTTGCCCTGTGATCTGAGACATTTGAACACTGAGCCAA
TGGAAATCTTCAGAAACTGTGTAAAGATTGAAGAAATCATGCCGAATGGTGACCCACTGT TGGCTGGCCAGAACACCGTGGATGAGGTTTACGTCTCCCGCCCCAGCCACTTCTACACTGA
TGGACCCTTGATGCCTTCTGACTTCTAGGAACCACATTTCCTCTGTTCTTTTCATATCTCTTG
CCCTTCCTACTCCTCATAGCATGATATTGTTCTCCAAGGATGGGAATCAGGCATGTGTCCCT
TCCAAGCTGTGTTAACTGTTCAAACTCAGGCCTGTGTGACTCCATTGGGGTGAGAGGTGAA
AGCATAACATGGGTACAGAGGGGACAACAATGAATCAGAACAGATGCTGAGCCATAGGT
CTAAATAGGATCCTGGAGGCTGCCTGCTGTGCTGGGAGGTATAGGGGTCCTGGGGGCAGG
CCAGGGCAGTTGACAGGTACTTGGAGGGCTCAGGGCAGTGGCTTCTTTCCAGTATGGAAG
GATTTCAACATTTTAATAGTTGGTTAGGCTAAACTGGTGCATACTGGCATTGGCCCTTGGT
GGGGAGCACAGACACAGGATAGGACTCCATTTCTTTCTTCCATTCCTTCATGTCTAGGATA
ACTTGCTTTCTTCTTTCCTTTACTCCTGGCTCAAGCCCTGAATTTCTTCTTTTCCTGCAGGGG
TTGAGAGCTTTCTGCCTTAGCCTACCATGTGAAACTCTACCCTGAAGAAAGGGATGGATAG
GTTCCTCCCCCATAAAATGATCCTGCCAATCTAATGTGAGTGTGAAGCTTTGCACACTAGT
TTATGCTACCTAGTCTCCACTTTCTCAATGCTTAGGAGACAGATCACTCCTGGAGGCTGGG
GATGGTAGGATTGCTGGGGATTTTTTTTTTTTTAAACAGGGTCTCACTCTGTTGCCCAGGCT
AGAGTGCAATGGTGCAATCACAGCTCACTGCAGCCTCAACCTCCTGGGTTCAAGCAATCCT
CCTACCTCAGCCTCCTGGGTAGCTAGCACCATGGCATGCGCCACCATGCCCTATTTTTTTTT
TTTAAAGACAGGGTCTTGCTATATTGCCCAGGCTGGTCTTGAACTGGGCTCAAGTGATCCT
CACGCCTTGGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACTGTGCTTGGCCAGGAT
TGATCTCGGCTCACTGCAACCTCCGCCTTCCGGGTTCAAGTGACTCTCCTGCCTCAGCCTCC
GACGGGGTTTCTCCATGTTGGTCAGGCTGGTCTCGAACTCCTGACCTCAAGTGATCTGTCC ACCTCGGCCTCCCAGAGTGCTGGGATTACAGGCGTGAGCCACTGTTCCCAGCAGGAATTTC
CTTTCTCTATTATGTTATCATCCTCCCTTTTTTGTACAATATGTTGTTTACCTGAAAGGAAG GTTTCTATTCGTTGGTTGTGGACCTGGACAAAGTCCAAGTCTGTGGAACTTAAAACCTTGA AGGTCTGTCATAGGACTCTGGACAATCTCACACCTTAGCTATTCCCAGGGAACCCCAGGGG GCAACTGACATTGCTCCAAGATGTTCTCCTGATGTAGCTTGAGATATAAAGGAAAGGCCCT GCACAGGTGGCTGTTTCTTGTCTGTTATGTCAGAGGAACAGTCCTGTTCAGAAAGGGGCTC TTCTGAGCAGAAATGGCTAATAAACTTTGTGCTGATCTGGAAAAAAAAAAAAAAAAAAAA AAAAAAAA (SEQ ID NO: 57,974)
"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.
[00107] The IRF1 polypeptide sequence is:
[00108] MPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRSWAIHTG RYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRMLPPLTKNQRKERKS KSSRDAKSKAKRKSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGYMQDLEVEQALTPALSPCAVS STLPDWHIPVEWPDSTSDLYNFQVSPMPSTSEATTDEDEEGKLPEDIMKLLEQSEWQPTNVDG KGYLLNEPGVQPTSVYGDFSCKEEPEIDSPGGDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPS IQAIPCAP (SEQ ID NO: 57,975)
[00109] The IRFl nucleic acid sequence is:
[00110] AGAGCTCGCCACTCCTTAGTCGAGGCAAGACGTGCGCCCGAGCCCCGCCGAACCGAGGCC ACCCGGAGCCGTGCCCAGTCCACGCCGGCCGTGCCCGGCGGCCTTAAGAACCCGGCAACC TCTGCCTTCTTCCCTCTTCCACTCGGAGTCGCGCTCCGCGCGCCCTCACTGCAGCCCCTGCG TCGCCGGGACCCTCGCGCGCGACCGCCGAATCGCTCCTGCAGCAGAGCCAACATGCCCAT CACTCGGATGCGCATGAGACCCTGGCTAGAGATGCAGATTAATTCCAACCAAATCCCGGG GCTCATCTGGATTAATAAAGAGGAGATGATCTTCCAGATCCCATGGAAGCATGCTGCCAA GCATGGCTGGGACATCAACAAGGATGCCTGTTTGTTCCGGAGCTGGGCCATTCACACAGG CCGATACAAAGCAGGGGAAAAGGAGCCAGATCCCAAGACGTGGAAGGCCAACTTTCGCT GTGCCATGAACTCCCTGCCAGATATCGAGGAGGTGAAAGACCAGAGCAGGAACAAGGGC AGCTCAGCTGTGCGAGTGTACCGGATGCTTCCACCTCTCACCAAGAACCAGAGAAAAGAA AGAAAGTCGAAGTCCAGCCGAGATGCTAAGAGCAAGGCCAAGAGGAAGTCATGTGGGGA TTCCAGCCCTGATACCTTCTCTGATGGACTCAGCAGCTCCACTCTGCCTGATGACCACAGC AGCTACACAGTTCCAGGCTACATGCAGGACTTGGAGGTGGAGCAGGCCCTGACTCCAGCA CTGTCGCCATGTGCTGTCAGCAGCACTCTCCCCGACTGGCACATCCCAGTGGAAGTTGTGC CGGACAGCACCAGTGATCTGTACAACTTCCAGGTGTCACCCATGCCCTCCACCTCTGAAGC TACAACAGATGAGGATGAGGAAGGGAAATTACCTGAGGACATCATGAAGCTCTTGGAGCA GTCGGAGTGGCAGCCAACAAACGTGGATGGGAAGGGGTACCTACTCAATGAACCTGGAGT CCAGCCCACCTCTGTCTATGGAGACTTTAGCTGTAAGGAGGAGCCAGAAATTGACAGCCC AGGGGGGGATATTGGGCTGAGTCTACAGCGTGTCTTCACAGATCTGAAGAACATGGATGC CACCTGGCTGGACAGCCTGCTGACCCCAGTCCGGTTGCCCTCCATCCAGGCCATTCCCTGT GCACCGTAGCAGGGCCCCTGGGCCCCTCTTATTCCTCTAGGCAAGCAGGACCTGGCATCAT GGTGGATATGGTGCAGAGAAGCTGGACTTCTGTGGGCCCCTCAACAGCCAAGTGTGACCC CACTGCCAAGTGGGGATGGGGCCTCCCTCCTTGGGTCATTGACCTCTCAGGGCCTGGCAGG CCAGTGTCTGGGTTTTTCTTGTGGTGTAAAGCTGGCCCTGCCTCCTGGGAAGATGAGGTTC
GCCCAGCTGCCTGGAGAGGGTCTCGCTGTCACTGGCTGGCTCCTAGGGGAACAGACCAGT GACCCCAGAAAAGCATAACACCAATCCCAGGGCTGGCTCTGCACTAAGAGAAAATTGCAC TAAATGAATCTCGTTCCCAAAGAACTACCCCCTTTTCAGCTGAGCCCTGGGGACTGTTCCA AAGCCAGTGAAATGTGAAGGAAAGTGGGGTCCTTCGGGGCGATGCTCCCTCAGCCTCAGA
CGTGTGGATCTTGCCACATTTCTGATCAGAGGTGTACACTAACATTTCCCCCGAGCTCTTG GCCTTTGCATTTATTTATACAGTGCCTTGCTCGGCGCCCACCACCCCCTCAAGCCCCAGCA GCCCTCAACAGGCCCAGGGAGGGAAGTGTGAGCGCCTTGGTATGACTTAAAATTGGAAAT
Figure imgf000029_0001
TGACTTTGGGGTGAACAGGGACATGCATCTATTATAAAATCCTTTCGGCCAGGCGCGGTGG CTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGATGGGTGGATCACCTGAGGTCAGG AGTTCGAGACCAGCCTGGTGAAACTCCATTTCTACTAAAAATGCAAAAATTAGCTGGGCGT GGTTGCGGGTGCTTGTAATCCCAGCTACTCAGGAGGCTGAGGCAAGAGAATCGCTTGAAC CTGGGAGGTGGAGGTTGCAGTGAGCCGAGAACATGCCATTGCACTCCAGCCCGGGCACCA AAAAAAAAAAAAAAAAAAAAAACCTTTCATTTGGCCGGGCATGGTGGCTTATGCCTGTAA TCCTGGCACTTTGGGAGGCCAAGGTGGGCAGATCACCTGAGGTCAGGAGTTTGAGACCAG CCTGGCCAACATGGTGAAACCTCATCTCTACTAAAAATACAAAAATTAGGCCGGGCACGG TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCAGAGGCGGGCGGATCACGAGGTCAG GAGATCAAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATATAAAAAA TTAGCCGGGCCTAGTGGCGGGTGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAG AATGGCATGAACCCCGGAGGCAGAGCTTGCAGTGAGCCGAGATTGCACCACTGCACTACA GCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAATTAGCCGGGCCTG GTGGCGGGCGCCTGTAATCCCAGCTACTGTGGAGGCTGAAGCACAAGAATCACTTGAACC CGGGAGATGGAGGTTGCAGTGAGCTGAGACTGTGCCACTGCACTCCAGCCTGGGTGACAA GAGTGAGACTTTGTCTCAAAAAAAAAAAAATCCTTTTGTTTATGTTCACATAGACAATGGC AGAAGGAGGGGACATTCCTGTCATAGGAACATGCTTATATAAACATAGTCACCTGTCCTTG ACTATCACCAGGGCTGTCAGTTGATTCTGGGCTCCTGGGGCCCAAGGAGTGTTAAGTTTTG AGGCATGTGCCATAGGTGATGTGTCCTGCTAACACACAGATGCTGCTCCAAAAAGTCAGTT GATATGACACAGTCACAGACAGAACAGTCAGCAGCCCAAGAAAGGTCCTCACGGCTGCTG TGCTGGGTAGCACTTGCCATCCAGTTTCTAGAGTGATGAAATGCTCTGTCTGTACCGTTCA ATACAGTAGGCACTGGCACTAGCCACATGTGCCAGCTAAGCACTTGAAATGTGGCCAGTG CAATAAGGAATTGAACTTTTAATTGCATTTAATAAACTGTATGTAAATAGTCAAAAAAAAA AAAAA (SEQ ID NO: 57,976)
"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.
[00112] The TLR1 polypeptide sequence is:
[00113] MTSIFHFAIIFMLILQIRIQLSEESEFLVDRSKNGLIHVPKDLSQKTTILNISQNYISELWTSDILSLS KLRILIISHNRIQYLDISVFKFNQELEYLDLSHNKLVKISCHPTVNLKHLDLSFNAFDALPICKEF GNMSQLKFLGLSTTHLEKSSVLPIAHLNISKVLLVLGETYGEKEDPEGLQDFNTESLHIVFPTNK EFHFILDVSVKTVANLELSNIKCVLEDNKCSYFLSILAKLQTNPKLSNLTLNNIETTWNSFIRILQ LVWHTTVWYFSISNVKLQGQLDFRDFDYSGTSLKALSIHQWSDVFGFPQSYIYEIFSNMNIKN FTVSGTRMVHMLCPSKISPFLHLDFSNNLLTDTVFENCGHLTELETLILQMNQLKELSKIAEMT TQMKSLQQLDISQNSVSYDEKKGDCSWTKSLLSLNMSSNILTDTIFRCLPPRIKVLDLHSNKIKS IPKQWKLEALQELNVAFNSLTDLPGCGSFSSLSVLIIDHNSVSHPSADFFQSCQKMRSIKAGDN PFQCTCELGEFVKNIDQVSSEVLEGWPDSYKCDYPESYRGTLLKDFHMSELSCNITLLIVTIVAT MLVLAVTVTSLCSYLDLPWYLRMVCQWTQTRRRARNIPLEELQRNLQFHAFISYSGHDSFWV KNELLPNLEKEGMQICLHERNFVPGKSIVENIITCIEKSYKSIFVLSPNFVQSEWCHYELYFAHH NLFHEGSNSLILILLEPIPQYSIPSSYHKLKSLMARRTYLEWPKEKSKRGLFWANLRAAINIKLTE QAKK (SEQ ID NO: 57,977)
[00114] The TLR1 nucleic acid sequence is:
[00115] ACAGACTGCCAAATGGAACAGACAAGCAGGTTGTCTTGTGTTAAAGAAAATGAGATATAA GTCAGTTACTCCCGGAGGCAATGCTGCTGTTCAGCTCTTCTGTTTTTGTGGCCAGGGTCTTC ATGAACACTAATAGGGGTACCAGGCCCTCTTCCTCGTTAGAAGAAATCAGGATAACAAAG GCATATTGGGCACCCCTACAAAAGGAATCTGTATCTGTATCAAGATGATCTGAAGAACAG CTTCTACCTTTAGGAATGTCTAGTGTTCCAAAATGACTAGCATCTTCCATTTTGCCATTATC
GGTCAAAAAACGGTCTCATCCACGTTCCTAAAGACCTATCCCAGAAAACAACAATCTTAA
ATATATCGCAAAATTATATATCTGAGCTTTGGACTTCTGACATCTTATCACTGTCAAAACTG
AGGATTTTGATAATTTCTCATAATAGAATCCAGTATCTTGATATCAGTGTTTTCAAATTCAA
CCAGGAATTGGAATACTTGGATTTGTCCCACAACAAGTTGGTGAAGATTTCTTGCCACCCT
ACTGTGAACCTCAAGCACTTGGACCTGTCATTTAATGCATTTGATGCCCTGCCTATATGCA
AAGAGTTTGGCAATATGTCTCAACTAAAATTTCTGGGGTTGAGCACCACACACTTAGAAAA
ATCTAGTGTGCTGCCAATTGCTCATTTGAATATCAGCAAGGTCTTGCTGGTCTTAGGAGAG
ACTTATGGGGAAAAAGAAGACCCTGAGGGCCTTCAAGACTTTAACACTGAGAGTCTGCAC
ATTGTGTTCCCCACAAACAAAGAATTCCATTTTATTTTGGATGTGTCAGTCAAGACTGTAG
CAAATCTGGAACTATCTAATATCAAATGTGTGCTAGAAGATAACAAATGTTCTTACTTCCT
AAGTATTCTGGCGAAACTTCAAACAAATCCAAAGTTATCAAATCTTACCTTAAACAACATT
GAAACAACTTGGAATTCTTTCATTAGGATCCTCCAGCTGGTTTGGCATACAACTGTATGGT ATTTCTCAATTTCAAACGTGAAGCTACAGGGTCAGCTGGACTTCAGAGATTTTGATTATTC
TGGCACTTCCTTGAAGGCCTTGTCTATACACCAAGTTGTCAGCGATGTGTTCGGTTTTCCGC
AAAGTTATATCTATGAAATCTTTTCGAATATGAACATCAAAAATTTCACAGTGTCTGGTAC
ACGCATGGTCCACATGCTTTGCCCATCCAAAATTAGCCCGTTCCTGCATTTGGATTTTTCCA
ATAATCTCTTAACAGACACGGTTTTTGAAAATTGTGGGCACCTTACTGAGTTGGAGACACT
TATTTTACAAATGAATCAATTAAAAGAACTTTCAAAAATAGCTGAAATGACTACACAGAT
GAAGTCTCTGCAACAATTGGATATTAGCCAGAATTCTGTAAGCTATGATGAAAAGAAAGG
AGACTGTTCTTGGACTAAAAGTTTATTAAGTTTAAATATGTCTTCAAATATACTTACTGACA
CTATTTTCAGATGTTTACCTCCCAGGATCAAGGTACTTGATCTTCACAGCAATAAAATAAA
GAGCATTCCTAAACAAGTCGTAAAACTGGAAGCTTTGCAAGAACTCAATGTTGCTTTCAAT
TCTTTAACTGACCTTCCTGGATGTGGCAGCTTTAGCAGCCTTTCTGTATTGATCATTGATCA
CAATTCAGTTTCCCACCCATCGGCTGATTTCTTCCAGAGCTGCCAGAAGATGAGGTCAATA
AAAGCAGGGGACAATCCATTCCAATGTACCTGTGAGCTAGGAGAATTTGTCAAAAATATA
GACCAAGTATCAAGTGAAGTGTTAGAGGGCTGGCCTGATTCTTATAAGTGTGACTACCCGG
AAAGTTATAGAGGAACCCTACTAAAGGACTTTCACATGTCTGAATTATCCTGCAACATAAC
TCTGCTGATCGTCACCATCGTTGCCACCATGCTGGTGTTGGCTGTGACTGTGACCTCCCTCT
GCAGCTACTTGGATCTGCCCTGGTATCTCAGGATGGTGTGCCAGTGGACCCAGACCCGGCG
CAGGGCCAGGAACATACCCTTAGAAGAACTCCAAAGAAATCTCCAGTTTCATGCATTTATT
TCATATAGTGGGCACGATTCTTTCTGGGTGAAGAATGAATTATTGCCAAACCTAGAGAAAG
AAGGTATGCAGATTTGCCTTCATGAGAGAAACTTTGTTCCTGGCAAGAGCATTGTGGAAAA
TATCATCACCTGCATTGAGAAGAGTTACAAGTCCATCTTTGTTTTGTCTCCCAACTTTGTCC
AGAGTGAATGGTGCCATTATGAACTCTACTTTGCCCATCACAATCTCTTTCATGAAGGATC
TAATAGCTTAATCCTGATCTTGCTGGAACCCATTCCGCAGTACTCCATTCCTAGCAGTTATC
ACAAGCTCAAAAGTCTCATGGCCAGGAGGACTTATTTGGAATGGCCCAAGGAAAAGAGCA
AAAGAAATAGATTACACATCAAGTGAAAAATATTCCTCCTGTTGATATTGCTGCTTTTGGA AGTTCCAACAATGACTTTATTTTGCATCAGCATAGATGTAAACACAATTGTGAGTGTATGA TGTAGGTAAAAATATATACCTTCGGGTCGCAGTTCACCATTTATATGTGGTATTAAAAATT AATGAAATGATATAACTTTGATTTAAACAGTTCTGACACATAAAAAAAAAAAAAAAAAA
(SEQ ID NO: 57,978)
"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:
[00118] MPHTLWMVWVLGVIISLSKEESSNQASLSCDRNGICKGSSGSLNSIPSGLTEAVKSLDLSN RIT YISNSDLQRCVNLQALVLTSNGINTIEEDSFSSLGSLEHLDLSYNYLSNLSSSWFKPLSSLTFLNL LGNPYKTLGETSLFSHLTKLQILRVGNMDTFTKIQRKDFAGLTFLEELEIDASDLQSYEPKSLKS IQNVSHLILHMKQHILLLEIFVDVTSSVECLELRDTDLDTFHFSELSTGETNSLIKKFTFRNVKIT DESLFQVMKLLNQISGLLELEFDDCTLNGVGNFRASDNDRVIDPGKVETLTIRRLHIPRFYLFYD LSTLYSLTERVKRITVENSKVFLVPCLLSQHLKSLEYLDLSENLMVEEYLKNSACEDAWPSLQT LILRQNHLASLEKTGETLLTLKNLTNIDISK SFHSMPETCQWPEKMKYLNLSSTRIHSVTGCIP KTLEILDVSN LNLFSLNLPQLKELYISRNKLMTLPDASLLPMLLVLKISRNAITTFSKEQLDSF HTLKTLEAGGN FICSCEFLSFTQEQQALAKVLIDWPANYLCDSPSHVRGQQVQDVRLSVSEC HRTALVSGMCCALFLLILLTGVLCHRFHGLWYMKMMWAWLQAKRKPRKAPSRNICYDAFVS YSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSE WCKYELDFSHFRLFDEN DAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFW VNLRAAIKS (SEQ ID NO: 57,979)
[00119] The TLR2 nucleic acid sequence is:
[00120] CGGAGGCAGCGAGAAAGCGCAGCCAGGCGGCTGCTCGGCGTTCTCTCAGGTGACTGCTCG GAGTTCTCCCAGTGTTTGGTGTTGCAAGCAGGATCCAAAGGAGACCTATAGTGACTCCCAG GAGCTCTTAGTGACCAAGTGAAGGTACCTGTGGGGCTCATTGTGCCCATTGCTCTTTCACT GCTTTCAACTGGTAGTTGTGGGTTGAAGCACTGGACAATGCCACATACTTTGTGGATGGTG TGGGTCTTGGGGGTCATCATCAGCCTCTCCAAGGAAGAATCCTCCAATCAGGCTTCTCTGT CTTGTGACCGCAATGGTATCTGCAAGGGCAGCTCAGGATCTTTAAACTCCATTCCCTCAGG GCTCACAGAAGCTGTAAAAAGCCTTGACCTGTCCAACAACAGGATCACCTACATTAGCAA CAGTGACCTACAGAGGTGTGTGAACCTCCAGGCTCTGGTGCTGACATCCAATGGAATTAAC ACAATAGAGGAAGATTCTTTTTCTTCCCTGGGCAGTCTTGAACATTTAGACTTATCCTATAA TTACTTATCTAATTTATCGTCTTCCTGGTTCAAGCCCCTTTCTTCTTTAACATTCTTAAACTT
CAAATCCTGAGAGTGGGAAATATGGACACCTTCACTAAGATTCAAAGAAAAGATTTTGCT
GGACTTACCTTCCTTGAGGAACTTGAGATTGATGCTTCAGATCTACAGAGCTATGAGCCAA
AAAGTTTGAAGTCAATTCAGAATGTAAGTCATCTGATCCTTCATATGAAGCAGCATATTTT
GATTTGGACACTTTCCATTTTTCAGAACTATCCACTGGTGAAACAAATTCATTGATTAAAA AGTTTACATTTAGAAATGTGAAAATCACCGATGAAAGTTTGTTTCAGGTTATGAAACTTTT GAATCAGATTTCTGGATTGTTAGAATTAGAGTTTGATGACTGTACCCTTAATGGAGTTGGT AATTTTAGAGCATCTGATAATGACAGAGTTATAGATCCAGGTAAAGTGGAAACGTTAACA ATCCGGAGGCTGCATATTCCAAGGTTTTACTTATTTTATGATCTGAGCACTTTATATTCACT
CTTTCACAACATTTAAAATCATTAGAATACTTGGATCTCAGTGAAAATTTGATGGTTGAAG AATACTTGAAAAATTCAGCCTGTGAGGATGCCTGGCCCTCTCTACAAACTTTAATTTTAAG
GCAAAATCATTTGGCATCATTGGAAAAAACCGGAGAGACTTTGCTCACTCTGAAAAACTT
GACTAACATTGATATCAGTAAGAATAGTTTTCATTCTATGCCTGAAACTTGTCAGTGGCCA
GAAAAGATGAAATATTTGAACTTATCCAGCACACGAATACACAGTGTAACAGGCTGCATT
CCCAAGACACTGGAAATTTTAGATGTTAGCAACAACAATCTCAATTTATTTTCTTTGAATTT
GCCGCAACTCAAAGAACTTTATATTTCCAGAAATAAGTTGATGACTCTACCAGATGCCTCC
CTCTTACCCATGTTACTAGTATTGAAAATCAGTAGGAATGCAATAACTACGTTTTCTAAGG
AGCAACTTGACTCATTTCACACACTGAAGACTTTGGAAGCTGGTGGCAATAACTTCATTTG
CTCCTGTGAATTCCTCTCCTTCACTCAGGAGCAGCAAGCACTGGCCAAAGTCTTGATTGAT
TGGCCAGCAAATTACCTGTGTGACTCTCCATCCCATGTGCGTGGCCAGCAGGTTCAGGATG
TCCGCCTCTCGGTGTCGGAATGTCACAGGACAGCACTGGTGTCTGGCATGTGCTGTGCTCT
GTTCCTGCTGATCCTGCTCACGGGGGTCCTGTGCCACCGTTTCCATGGCCTGTGGTATATGA
AAATGATGTGGGCCTGGCTCCAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAAC
ATCTGCTATGATGCATTTGTTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAGAACCTTA
TGGTCCAGGAGCTGGAGAACTTCAATCCCCCCTTCAAGTTGTGTCTTCATAAGCGGGACTT
CATTCCTGGCAAGTGGATCATTGACAATATCATTGACTCCATTGAAAAGAGCCACAAAACT
GTCTTTGTGCTTTCTGAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTCT
CCCATTTCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTTCTGGAGCCCATT
GAGAAAAAAGCCATTCCCCAGCGCTTCTGCAAGCTGCGGAAGATAATGAACACCAAGACC
TACCTGGAGTGGCCCATGGACGAGGCTCAGCGGGAAGGATTTTGGGTAAATCTGAGAGCT
GCGATAAAGTCCTAGGTTCCCATATTTAAGACCAGTCTTTGTCTAGTTGGGATCTTTATGTC
ACTAGTTATAGTTAAGTTCATTCAGACATAATTATATAAAAACTACGTGGATGTACCGTCA
TTTGAGGACTTGCTTACTAAAACTACAAAACTTCAAATTTTGTCTGGGGTGCTGTTTTATAA
ATAAGTCTATTACTGATATCTGAATATAGTCCCTTGGTATCCAAGGGAATTGGTTGCAGGA TCCTCGTGGATATCAAAATTCATAGATGATCAAGTCCCTTATAAGAGTGGCATAGTATTTG
Figure imgf000033_0001
AGAACCCATGGATATAGAGGGCCAACTGTAATCTGTAGCAACTGGCTTAGTTCATTAGGA
AACAGCACAAATGAACTTAAGATTCTCAATGACTGTGTCATTCTTTCTTCCTGCTAAGAGA
CTCCTCTGTGGCCACAAAAGGCATTCTCTGTCCTACCTAGCTGTCACTTCTCTGTGCAGCTG
ATCTCAAGAGCAACAAGGCAAAGTATTTGGGGCACTCCCCAAAACTTGTTGCTATTCCTAG
AAAAAAGTGCTGTGTATTTCCTATTAAACTTTACAGGATGAGAAAAAAAAAAAAAAAAA
(SEQ ID NO: 57,980)
"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.
[00122] The TLR3 polypeptide sequence is:
[00123] MRQTLPCIYFWGGLLPFGMLCASSTTKCTVSHEVADCSHLKLTQVPDDLPTNITVLNLTHNQL RRLPAANFTRYSQLTSLDVGFNTISKLEPELCQKLPMLKVLNLQHNELSQLSDKTFAFCTNLTE LHLMSNSIQKIKNNPFVKQKNLITLDLSHNGLSSTKLGTQVQLENLQELLLSNNKIQALKSEEL DIFANSSLKKLELSSNQIKEFSPGCFHAIGRLFGLFLNNVQLGPSLTEKLCLELANTSIRNLSLSNS QLSTTSNTTFLGLKWTNLTMLDLSYNNLNWGNDSFAWLPQLEYFFLEYNNIQHLFSHSLHGL FNVRYLNLKRSFTKQSISLASLPKIDDFSFQWLKCLEHLNMEDNDIPGIKSNMFTGLINLKYLSL SNSFTSLRTLTNETFVSLAHSPLHILNLTKNKISKIESDAFSWLGHLEVLDLGLNEIGQELTGQE WRGLENIFEIYLSYNKYLQLTRNSFALVPSLQRLMLRRVALKNVDSSPSPFQPLRNLTILDLSNN NIANINDDMLEGLEKLEILDLQHNNLARLWKHANPGGPIYFLKGLSHLHILNLESNGFDEIPVE VFKDLFELKIIDLGLNNLNTLPASVFNNQVSLKSLNLQKNLITSVEKKVFGPAFRNLTELDMRF NPFDCTCESIAWFVNWINETHTNIPELSSHYLCNTPPHYHGFPVRLFDTSSCKDSAPFELFFMIN TSILLIFIFIVLLIHFEGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHF SSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQA IEQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSV
H (SEQ ID NO: 57,981)
[00124] The TLR3 nucleic acid sequence is:
[00125] CACTTTCGAGAGTGCCGTCTATTTGCCACACACTTCCCTGATGAAATGTCTGGATTTGGACT AAAGAAAAAAGGAAAGGCTAGCAGTCATCCAACAGAATCATGAGACAGACTTTGCCTTGT ATCTACTTTTGGGGGGGCCTTTTGCCCTTTGGGATGCTGTGTGCATCCTCCACCACCAAGTG CACTGTTAGCCATGAAGTTGCTGACTGCAGCCACCTGAAGTTGACTCAGGTACCCGATGAT CTACCCACAAACATAACAGTGTTGAACCTTACCCATAATCAACTCAGAAGATTACCAGCCG CCAACTTCACAAGGTATAGCCAGCTAACTAGCTTGGATGTAGGATTTAACACCATCTCAAA ACTGGAGCCAGAATTGTGCCAGAAACTTCCCATGTTAAAAGTTTTGAACCTCCAGCACAAT GAGCTATCTCAACTTTCTGATAAAACCTTTGCCTTCTGCACGAATTTGACTGAACTCCATCT CATGTCCAACTCAATCCAGAAAATTAAAAATAATCCCTTTGTCAAGCAGAAGAATTTAATC ACATTAGATCTGTCTCATAATGGCTTGTCATCTACAAAATTAGGAACTCAGGTTCAGCTGG AAAATCTCCAAGAGCTTCTATTATCAAACAATAAAATTCAAGCGCTAAAAAGTGAAGAAC TGGATATCTTTGCCAATTCATCTTTAAAAAAATTAGAGTTGTCATCGAATCAAATTAAAGA GTTTTCTCCAGGGTGTTTTCACGCAATTGGAAGATTATTTGGCCTCTTTCTGAACAATGTCC AGCTGGGTCCCAGCCTTACAGAGAAGCTATGTTTGGAATTAGCAAACACAAGCATTCGGA ATCTGTCTCTGAGTAACAGCCAGCTGTCCACCACCAGCAATACAACTTTCTTGGGACTAAA GTGGACAAATCTCACTATGCTCGATCTTTCCTACAACAACTTAAATGTGGTTGGTAACGAT TCCTTTGCTTGGCTTCCACAACTAGAATATTTCTTCCTAGAGTATAATAATATACAGCATTT GTTTTCTCACTCTTTGCACGGGCTTTTCAATGTGAGGTACCTGAATTTGAAACGGTCTTTTA
AAATGTTTGGAGCACCTTAACATGGAAGATAATGATATTCCAGGCATAAAAAGCAATATG
TTCACAGGATTGATAAACCTGAAATACTTAAGTCTATCCAACTCCTTTACAAGTTTGCGAA
CTTTGACAAATGAAACATTTGTATCACTTGCTCATTCTCCCTTACACATACTCAACCTAACC
AAGAATAAAATCTCAAAAATAGAGAGTGATGCTTTCTCTTGGTTGGGCCACCTAGAAGTA
CTTGACCTGGGCCTTAATGAAATTGGGCAAGAACTCACAGGCCAGGAATGGAGAGGTCTA
GAAAATATTTTCGAAATCTATCTTTCCTACAACAAGTACCTGCAGCTGACTAGGAACTCCT
TTGCCTTGGTCCCAAGCCTTCAACGACTGATGCTCCGAAGGGTGGCCCTTAAAAATGTGGA
TAGCTCTCCTTCACCATTCCAGCCTCTTCGTAACTTGACCATTCTGGATCTAAGCAACAACA
ACATAGCCAACATAAATGATGACATGTTGGAGGGTCTTGAGAAACTAGAAATTCTCGATTT
GCAGCATAACAACTTAGCACGGCTCTGGAAACACGCAAACCCTGGTGGTCCCATTTATTTC
CTAAAGGGTCTGTCTCACCTCCACATCCTTAACTTGGAGTCCAACGGCTTTGACGAGATCC
CAGTTGAGGTCTTCAAGGATTTATTTGAACTAAAGATCATCGATTTAGGATTGAATAATTT
AAACACACTTCCAGCATCTGTCTTTAATAATCAGGTGTCTCTAAAGTCATTGAACCTTCAG
AAGAATCTCATAACATCCGTTGAGAAGAAGGTTTTCGGGCCAGCTTTCAGGAACCTGACTG
AGTTAGATATGCGCTTTAATCCCTTTGATTGCACGTGTGAAAGTATTGCCTGGTTTGTTAAT
TGGATTAACGAGACCCATACCAACATCCCTGAGCTGTCAAGCCACTACCTTTGCAACACTC
CACCTCACTATCATGGGTTCCCAGTGAGACTTTTTGATACATCATCTTGCAAAGACAGTGC
TCTCATCCACTTTGAGGGCTGGAGGATATCTTTTTATTGGAATGTTTCAGTACATCGAGTTC
TTGGTTTCAAAGAAATAGACAGACAGACAGAACAGTTTGAATATGCAGCATATATAATTC
ATGCCTATAAAGATAAGGATTGGGTCTGGGAACATTTCTCTTCAATGGAAAAGGAAGACC
AATCTCTCAAATTTTGTCTGGAAGAAAGGGACTTTGAGGCGGGTGTTTTTGAACTAGAAGC
AATTGTTAACAGCATCAAAAGAAGCAGAAAAATTATTTTTGTTATAACACACCATCTATTA
AAAGACCCATTATGCAAAAGATTCAAGGTACATCATGCAGTTCAACAAGCTATTGAACAA
AATCTGGATTCCATTATATTGGTTTTCCTTGAGGAGATTCCAGATTATAAACTGAACCATG
CACTCTGTTTGCGAAGAGGAATGTTTAAATCTCACTGCATCTTGAACTGGCCAGTTCAGAA
AGAACGGATAGGTGCCTTTCGTCATAAATTGCAAGTAGCACTTGGATCCAAAAACTCTGTA
CATTAAATTTATTTAAATATTCAATTAGCAAAGGAGAAACTTTCTCAATTTAAAAAGTTCT
ATGGCAAATTTAAGTTTTCCATAAAGGTGTTATAATTTGTTTATTCATATTTGTAAATGATT
ATATTCTATCACAATTACATCTCTTCTAGGAAAATGTGTCTCCTTATTTCAGGCCTATTTTT
GACAATTGACTTAATTTTACCCAAAATAAAACATATAAGCACGTAAAAAAAAAAAAAAAA
AA (SEQ ID NO: 57,982) [00126] "TNF" or "tumor necrosis factor" 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: 57,983; 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.
[00127] The TNF polypeptide sequence is:
[00128] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQPvEEFPR DLSLISPLAQAVRSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLWP SEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYE PIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 57,983)
[00129] The TNF nucleic acid sequence is:
[00130] CAGACGCTCCCTCAGCAAGGACAGCAGAGGACCAGCTAAGAGGGAGAGAAGCAACTACA GACCCCCCCTGAAAACAACCCTCAGACGCCACATCCCCTGACAAGCTGCCAGGCAGGTTC TCTTCCTCTCACATACTGACCCACGGCTCCACCCTCTCTCCCCTGGAAAGGACACCATGAG CACTGAAAGCATGATCCGGGACGTGGAGCTGGCCGAGGAGGCGCTCCCCAAGAAGACAG GGGGGCCCCAGGGCTCCAGGCGGTGCTTGTTCCTCAGCCTCTTCTCCTTCCTGATCGTGGC AGGCGCCACCACGCTCTTCTGCCTGCTGCACTTTGGAGTGATCGGCCCCCAGAGGGAAGA GTTCCCCAGGGACCTCTCTCTAATCAGCCCTCTGGCCCAGGCAGTCAGATCATCTTCTCGA ACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAACCCTCAAGCTGAGGGGCAGCTC CAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCTGAGAGATAAC CAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCC AAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGCCGTCTCCTA CCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGAGACCCCAGA GGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCAGCTGGA GAAGGGTGACCGACTCAGCGCTGAGATCAATCGGCCCGACTATCTCGACTTTGCCGAGTCT GGGCAGGTCTACTTTGGGATCATTGCCCTGTGAGGAGGACGAACATCCAACCTTCCCAAAC GCCTCCCCTGCCCCAATCCCTTTATTACCCCCTCCTTCAGACACCCTCAACCTCTTCTGGCT CAAAAAGAGAATTGGGGGCTTAGGGTCGGAACCCAAGCTTAGAACTTTAAGCAACAAGAC CACCACTTCGAAACCTGGGATTCAGGAATGTGTGGCCTGCACAGTGAAGTGCTGGCAACC ACTAAGAATTCAAACTGGGGCCTCCAGAACTCACTGGGGCCTACAGCTTTGATCCCTGACA TCTGGAATCTGGAGACCAGGGAGCCTTTGGTTCTGGCCAGAATGCTGCAGGACTTGAGAA GACCTCACCTAGAAATTGACACAAGTGGACCTTAGGCCTTCCTCTCTCCAGATGTTTCCAG ACTTCCTTGAGACACGGAGCCCAGCCCTCCCCATGGAGCCAGCTCCCTCTATTTATGTTTG CACTTGTGATTATTTATTATTTATTTATTATTTATTTATTTACAGATGAATGTATTTATTTGG GAGACCGGGGTATCCTGGGGGACCCAATGTAGGAGCTGCCTTGGCTCAGACATGTTTTCCG TGAAAACGGAGCTGAACAATAGGCTGTTCCCATGTAGCCCCCTGGCCTCTGTGCCTTCTTT TGATTATGTTTTTTAAAATATTTATCTGATTAAGTTGTCTAAACAATGCTGATTTGGTGACC AACTGTCACTCATTGCTGAGCCTCTGCTCCCCAGGGGAGTTGTGTCTGTAATCGCCCTACT ATTCAGTGGCGAGAAATAAAGTTTGCTTAGAAAAGAAAAAAAAAAAAA (SEQ ID NO: 57,984)
[00131] "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).
[00132] "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.
[00133] The HLA-DQAl polypeptide sequence is:
[00134] MILNKALLLGALALTTVMSPCGGEDIVADHVASCGVNLYQFYGPSGQYTHEFDGDEQFYVDL ERKETAWRWPEFSKFGGFDPQGAL1WMAVAKHNLNIMIKRYNSTAATNEVPEVTVFSKSPVT LGQPNTLICLVDNIFPPVVNITWLSNGQSVTEGVSETSFLSKSDHSFFKISYLTFLPSADEIYDCK VEHWGLDQPLLKHWEPEIPAPMSELTETVVCALGLSVGLMGIWGTVFIIQGLRSVGASRHQG PL (SEQ ID NO: 57,985)
[00135] The HLA-DQAl nucleic acid sequence is:
[00136] ACAATTACTCTACAGCTCAGAACACCAACTGCTGAGGCTGCCTTGGGAAGAGGATGATCC TAAACAAAGCTCTGCTGCTGGGGGCCCTCGCTCTGACCACCGTGATGAGCCCCTGTGGAGG TGAAGACATTGTGGCTGACCACGTTGCCTCTTGTGGTGTAAACTTGTACCAGTTTTACGGT CCCTCTGGCCAGTACACCCATGAATTTGATGGAGATGAGCAGTTCTACGTGGACCTGGAGA GGAAGGAGACTGCCTGGCGGTGGCCTGAGTTCAGCAAATTTGGAGGTTTTGACCCGCAGG GTGCACTGAGAAACATGGCTGTGGCAAAACACAACTTGAACATCATGATTAAACGCTACA ACTCTACCGCTGCTACCAATGAGGTTCCTGAGGTCACAGTGTTTTCCAAGTCTCCCGTGAC ACTGGGTCAGCCCAACACCCTCATTTGTCTTGTGGACAACATCTTTCCTCCTGTGGTCAACA TCACATGGCTGAGCAATGGGCAGTCAGTCACAGAAGGTGTTTCTGAGACCAGCTTCCTCTC CAAGAGTGATCATTCCTTCTTCAAGATCAGTTACCTCACCTTCCTCCCTTCTGCTGATGAGA TTTATGACTGCAAGGTGGAGCACTGGGGCCTGGACCAGCCTCTTCTGAAACACTGGGAGC CTGAGATTCCAGCCCCTATGTCAGAGCTCACAGAGACTGTGGTCTGTGCCCTGGGGTTGTC TGTGGGCCTCATGGGCATTGTGGTGGGCACTGTCTTCATCATCCAAGGCCTGCGTTCAGTT GGTGCTTCCAGACACCAAGGGCCATTGTGAATCCCATCCTGGAAGGGAAGGTGCATCGCC ATCTACAGGAGCAGAAGAATGGACTTGCTAAATGACCTAGCACTATTCTCTGGCCCGATTT ATCATATCCCTTTTCTCCTCCAAATATTTCTCCTCTCACCTTTTCTCTGGGACTTAAGCTGCT
TTTTCTCAAATGTTACCTACAAAGACATGCCTGGGGTAAGCCACCCGGCTACCTAATTCCT CAGTAACCTCCATCTAAAATCTCCAAGGAAGCAATAAATTCCTTTTATGAGATCTATGTCA AATTTTTCCATCTTTCATCCAGGGCTGACTGAAACTATGGCTAATAATTGGGGTACTCTTAT
CCAAGTACAGTATAGCCTGATAATATGTTGATTTCTTAGCTGACATTAATATTTCTTGCTTC CTTGTGTTCCCACCCTTGGCACTGCCACCCACCCCTCAATTCAGGCAACAATGAAATTAAT GGATACCGTCTGCCCTTGGCCCAGAATTGTTATAGCAAAAATTTTAGAACCAAAAAATAA GTCTGTACTAATTTCAATGTGGCTTTTAAAAGTATGACAGAGAAATAAGTTAGGATAAAGG AAATTTGAATCTCA (SEQ ID NO: 57,986)
[00137] "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: 57,989-57,991.
[00138] The HLA-DQB 1 (isoform 1) polypeptide sequence is:
[00139] MSWKXALRIPGGLPvAATVTLMLSMLSTPVAEGRDSPEDFVYQFKGMCYFTNGTERVPvLVSRSI YNREEIVRFDSDVGEFRAVTLLGLPAAEYWNSQKDILERKRAAVDRVCRHNYQLELRTTLQR RVEPTVTISPSRTEALNHHNLLVCSVTDFYPAQIKVRWFRNDQEETAGWSTPLIRNGDWTFQI LVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSKMLSGIGGFVLGLIFLGLGLIIHH RSQKGLLH (SEQ ID NO: 57,987)
[00140] The HLA-DQB 1 (isoform 2) polypeptide sequence is:
[00141] MSWKXALRIPGDLRVATVTLMLAMLSSLLAEGRDSPEDFVFQFKGMCYFTNGTERVRLVTRY IYNREEYARFDSDVGVYRAVTPQGRPDAEYWNSQKEVLEGTRAELDTVCRHNYEVAFRGILQ RRVEPTVTISPSRTEALNHHNLLVCSVTDFYPGQIKVRWFRNDQEETAGWSTPLIRNGDWTFQ ILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSKMLSGVGGFVLGLIFLGLGLIIR QRSQKGPQGPPPAGLLH (SEQ ID NO: 57,988)
[00142] The HLA-DQB 1 (variant 1) nucleic acid sequence is:
CTTTTCCCTTCGTCTCAGTTATGTCTTGGAAGAAGGCTTTGCGGATCCCCGGAGACCTTCGG GTAGCAACTGTCACCTTGATGCTGGCGATGCTGAGCTCCCTACTGGCTGAGGGCAGAGACT CTCCCGAGGATTTCGTGTTCCAGTTTAAGGGCATGTGCTACTTCACCAACGGGACGGAGCG CGTGCGTCTTGTGACCAGATACATCTATAACCGAGAGGAGTACGCGCGCTTCGACAGCGA
CGTGGGGGTGTACCGCGCGGTGACGCCGCAGGGGCGGCCTGATGCCGAGTACTGGAACAG
CCAGAAGGAAGTCCTGGAGGGGACCCGGGCGGAGTTGGACACGGTGTGCAGACACAACT
ACGAGGTGGCGTTCCGCGGGATCTTGCAGAGGAGAGTGGAGCCCACAGTGACCATCTCCC
CATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGGTCTGCTCGGTGACAGATTTCTA
TCCAGGCCAGATCAAAGTCCGGTGGTTTCGGAATGATCAGGAGGAGACAGCCGGCGTTGT
GTCCACCCCCCTTATTAGGAATGGTGACTGGACTTTCCAGATCCTGGTGATGCTGGAAATG
ACTCCCCAGCGTGGAGATGTCTACACCTGCCACGTGGAGCACCCCAGCCTCCAGAGCCCC
ATCACCGTGGAGTGGCGGGCTCAGTCTGAATCTGCCCAGAGCAAGATGCTGAGTGGCGTT
GGAGGCTTCGTGCTGGGGCTGATCTTCCTTGGGCTGGGCCTTATCATCCGTCAAAGGAGTC
AGAAAGGGCTTCTGCACTGACTCCTGAGACTATTTTAACTAGGATTGGTTATCACTCTTCT
GTGATGCCTGCTTATGCCTGCCCAGAATTCCCAGCTGCCTGTGTCAGCTTGTCCCCCTGAG
ATCAAAGTCCTACAGTGGCTGTCACGCAGCCACCAGGTCATCTCCTTTCATCCCCACCCCA
AGGCGCTGGCTGTGACTCTGCTTCCTGCACTGACCCAGAGCCTCTGCCTGTGCATGGCCAG
CTGCGTCTACTCAGGTCCCAAGGGGTTTCTGTTTCTATTCTTTCCTCAGACTGCTCAAGAGA
AGCACATGAAAAACATTACCTGACTTTAGAGCTTTTTTACATAATTAAACATGATCCTGAG
TTATCTGTATTCTGAACTTTCTTAATTGAGAAGAGGCAGGAAATCACTGCAGAATGAAGGA
ACATCCCTTGAGGTGACCCAGCAAACCTGTGGCCAGAAGGAGGATTGTACCTTGAAAAGA
CACTGAAAGCATTTTGGGGTGTGAAGTAAGGGTGGGCAGAGGAGGTAGAAAATAATTCAA
TTGTCGCATCATTCATGGTTCTTTAATACTGATGCTCAGTGCATTGGCCTTAGAATATCCCA
GCCTCTCTTCTGGTTTGGTGAGTGCTGTGTAAATAAGCATGGTAGAATTGTTTGGAGACAT
ATATAGTGATCCTTGGTCACTGGTGTTTCAAACATTCTGGAAAGTCACATCGATCAAGAAT
AAAAAAAAAAAAAAA (SEQ ID NO: 57,989)
The HLA-DQB1 (variant 2) nucleic acid sequence is:
CTTTTCCCTTCGTCTCAGTTATGTCTTGGAAGAAGGCTTTGCGGATCCCCGGAGACCTTCGG
GTAGCAACTGTCACCTTGATGCTGGCGATGCTGAGCTCCCTACTGGCTGAGGGCAGAGACT
CTCCCGAGGATTTCGTGTTCCAGTTTAAGGGCATGTGCTACTTCACCAACGGGACGGAGCG
CGTGCGTCTTGTGACCAGATACATCTATAACCGAGAGGAGTACGCGCGCTTCGACAGCGA
CGTGGGGGTGTACCGCGCGGTGACGCCGCAGGGGCGGCCTGATGCCGAGTACTGGAACAG
CCAGAAGGAAGTCCTGGAGGGGACCCGGGCGGAGTTGGACACGGTGTGCAGACACAACT
ACGAGGTGGCGTTCCGCGGGATCTTGCAGAGGAGAGTGGAGCCCACAGTGACCATCTCCC
CATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGGTCTGCTCGGTGACAGATTTCTA
TCCAGGCCAGATCAAAGTCCGGTGGTTTCGGAATGATCAGGAGGAGACAGCCGGCGTTGT
GTCCACCCCCCTTATTAGGAATGGTGACTGGACTTTCCAGATCCTGGTGATGCTGGAAATG
ACTCCCCAGCGTGGAGATGTCTACACCTGCCACGTGGAGCACCCCAGCCTCCAGAGCCCC ATCACCGTGGAGTGGCGGGCTCAGTCTGAATCTGCCCAGAGCAAGATGCTGAGTGGCGTT
GGAGGCTTCGTGCTGGGGCTGATCTTCCTTGGGCTGGGCCTTATCATCCGTCAAAGGAGTC
AGAAAGGACCTCAAGGGCCTCCACCAGCAGGGCTTCTGCACTGACTCCTGAGACTATTTTA
ACTAGGATTGGTTATCACTCTTCTGTGATGCCTGCTTATGCCTGCCCAGAATTCCCAGCTGC
CTGTGTCAGCTTGTCCCCCTGAGATCAAAGTCCTACAGTGGCTGTCACGCAGCCACCAGGT
CATCTCCTTTCATCCCCACCCCAAGGCGCTGGCTGTGACTCTGCTTCCTGCACTGACCCAGA
GCCTCTGCCTGTGCATGGCCAGCTGCGTCTACTCAGGTCCCAAGGGGTTTCTGTTTCTATTC
CATAATTAAACATGATCCTGAGTTATCTGTATTCTGAACTTTCTTAATTGAGAAGAGGCAG
GAAATCACTGCAGAATGAAGGAACATCCCTTGAGGTGACCCAGCAAACCTGTGGCCAGAA
GGAGGATTGTACCTTGAAAAGACACTGAAAGCATTTTGGGGTGTGAAGTAAGGGTGGGCA
GAGGAGGTAGAAAATAATTCAATTGTCGCATCATTCATGGTTCTTTAATACTGATGCTCAG
TGCATTGGCCTTAGAATATCCCAGCCTCTCTTCTGGTTTGGTGAGTGCTGTGTAAATAAGC
ATGGTAGAATTGTTTGGAGACATATATAGTGATCCTTGGTCACTGGTGTTTCAAACATTCT
ATACTATTTTTGAGTCTAAATGA (SEQ ID NO: 57,990)
The HLA-DQB1 (variant 3) nucleic acid sequence is:
TTCCCTTCGTCTCAATTATGTCTTGGAAAAAGGCTTTGCGGATCCCCGGAGGCCTTCGGGC
AGCAACTGTGACCTTGATGCTGTCGATGCTGAGCACCCCAGTGGCTGAGGGCAGAGACTC
TCCCGAGGATTTCGTGTACCAGTTTAAGGGCATGTGCTACTTCACCAACGGGACAGAGCGC
GTGCGTCTTGTGAGCAGAAGCATCTATAACCGAGAAGAGATCGTGCGCTTCGACAGCGAC
GTGGGGGAGTTCCGGGCGGTGACGCTGCTGGGGCTGCCTGCCGCCGAGTACTGGAACAGC
CAGAAGGACATCCTGGAGAGGAAACGGGCGGCGGTGGACAGGGTGTGCAGACACAACTA
CCAGTTGGAGCTCCGCACGACCTTGCAGCGGCGAGTGGAGCCCACAGTGACCATCTCCCC
ATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGGTCTGCTCGGTGACAGATTTCTAT
CCAGCCCAGATCAAAGTCCGGTGGTTTCGGAATGACCAGGAGGAGACAGCTGGCGTTGTG
TCCACCCCCCTTATTAGGAATGGTGACTGGACCTTCCAGATCCTGGTGATGCTGGAAATGA
CTCCCCAGCGTGGAGACGTCTACACCTGCCACGTGGAGCACCCCAGCCTCCAGAGCCCCAT
CACCGTGGAGTGGCGGGCTCAATCTGAATCTGCCCAGAGCAAGATGCTGAGTGGCATTGG
AGGCTTCGTGCTGGGGCTGATCTTCCTCGGGCTGGGCCTTATCATCCATCACAGGAGTCAG
AAAGGGCTCCTGCACTGACTCCTGAGACTATTTTAACTGGGATTGGTTATCACTTTTCTGTA
ACGCCTGCTTGTCCCTGCCCAGAATTCCCAGCTGTCTGTGTCAGCCTGTCCCCCTGAGATCA
GAGTCCTACAGTGGCTGTCACGCAGCCACCAGGTCATCTCCTTTCATCCCCACCTTGAGGC
GGATGGCTGTGACCCTACTTCCTGCACTGACCCACAGCCTCTGCCTGTGCACGGCCAGCTG
CATCTACTCAGGCCCCAAGGGGTTTCTGTTTCTATTCTCTCCTCAGACTGCTCAAGAGAAG ATCTGTATTCTGAACTTCCTTAATTGAGCAGAGGCAGGAAATCACTGCAGAATGAAGGAA
CATACCTTGAGGTGACCCAGCCAACCTGTGCCCAGAAGGAGGGTTGTACCTTGAAAAGAC
ACTGAAAGAATTTGGGGTGCAAAGTCATGGTGGGCAGAGGAGGTAGAAAATCAACTCAGT
TGTTGCATCATTCATGGTTCTTTCATATTGATGTTCAGTGCAGTGGCCTGAGAATATCCCAG
CCTCTCTTCTGGTTTGGTGAGTGCTATATAAGTAAACATGGTGGAATTGTTTGGGGGCAGA
TATAGTGACCCTTGGTCACTGGTGTTTCAAACATTCTGGCAAGTCACATCAATCAAGAATA
(SEQ ID NO: 57,991)
[00148] "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.
[00149] The MYD88 (isoform 1) polypeptide sequence is:
[00150] MPvPDPvAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRPvRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEED CQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQ EMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRLARRPRGGCRRMWWSDDYLQSK ECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP
(SEQ ID NO: 57,992)
[00151] The MYD88 (isoform 2) polypeptide sequence is:
[00152] MRPDRAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEED CQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQ EMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMWWSDDYLQSKECDFQTKF ALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 57,993)
[00153] The MYD88 (isoform 3) polypeptide sequence is:
[00154] MRPDRAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGHM PERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVWSIASELIEKRCRRMW WSDDYLQSKECDFQTKFALSLSPGAHQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWF WTRLAKALSLP (SEQ ID NO: 57,994)
[00155] The MYD88 (isoform 4) polypeptide sequence is: [00156] MRPDRAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEED CQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGAAGWWWLSLMITCRARNVT SRPNLHSASLQVPIRSD (SEQ ID NO: 57,995)
[00157] The MYD88 (isoform 5) polypeptide sequence is:
[00158] MRPDRAEAPGPPAMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTA LAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIGAA GWWWLSLMITCRARNVTSRPNLHSASLQVPIRSD(SEQ ID NO: 57,996)
[00159] The MYD88 (isoform 1) nucleic acid sequence is:
[00160] AGATTCCTACTTCTTACGCCCCCCACATCACCCGCCTCGAGACCTCAAGGGTAGAGGTGGG CACCCCCGCCTCCGCACTTTTGCTCGGGGCTCCAGATTGTAGGGCAGGGCGGCGCTTCTCG GAAAGCGAAAGCCGGCGGGGCGGGGCGGGTGCCGCAGGAGAAAGAGGAAGCGCTGGCA GACAATGCGACCCGACCGCGCTGAGGCTCCAGGACCGCCCGCCATGGCTGCAGGAGGTCC CGGCGCGGGGTCTGCGGCCCCGGTCTCCTCCACATCCTCCCTTCCCCTGGCTGCTCTCAAC ATGCGAGTGCGGCGCCGCCTGTCTCTGTTCTTGAACGTGCGGACACAGGTGGCGGCCGACT GGACCGCGCTGGCGGAGGAGATGGACTTTGAGTACTTGGAGATCCGGCAACTGGAGACAC AAGCGGACCCCACTGGCAGGCTGCTGGACGCCTGGCAGGGACGCCCTGGCGCCTCTGTAG GCCGACTGCTCGAGCTGCTTACCAAGCTGGGCCGCGACGACGTGCTGCTGGAGCTGGGAC CCAGCATTGAGGAGGATTGCCAAAAGTATATCTTGAAGCAGCAGCAGGAGGAGGCTGAGA AGCCTTTACAGGTGGCCGCTGTAGACAGCAGTGTCCCACGGACAGCAGAGCTGGCGGGCA TCACCACACTTGATGACCCCCTGGGGCATATGCCTGAGCGTTTCGATGCCTTCATCTGCTAT TGCCCCAGCGACATCCAGTTTGTGCAGGAGATGATCCGGCAACTGGAACAGACAAACTAT CGACTGAAGTTGTGTGTGTCTGACCGCGATGTCCTGCCTGGCACCTGTGTCTGGTCTATTGC TAGTGAGCTCATCGAAAAGAGGTTGGCTAGAAGGCCACGGGGTGGGTGCCGCCGGATGGT GGTGGTTGTCTCTGATGATTACCTGCAGAGCAAGGAATGTGACTTCCAGACCAAATTTGCA CTCAGCCTCTCTCCAGGTGCCCATCAGAAGCGACTGATCCCCATCAAGTACAAGGCAATGA AGAAAGAGTTCCCCAGCATCCTGAGGTTCATCACTGTCTGCGACTACACCAACCCCTGCAC CAAATCTTGGTTCTGGACTCGCCTTGCCAAGGCCTTGTCCCTGCCCTGAAGACTGTTCTGA GGCCCTGGGTGTGTGTGTATCTGTCTGCCTGTCCATGTACTTCTGCCCTGCCTCCTCCTTTC GTTGTAGGAGGAATCTGTGCTCTACTTACCTCTCAATTCCTGGAGATGCCAACTTCACAGA CACGTCTGCAGCAGCTGGACATCACATTTCATGTCCTGCATGGAACCAGTGGCTGTGAGTG GCATGTCCACTTGCTGGATTATCAGCCAGGACACTATAGAACAGGACCAGCTGAGACTAA GAAGGACCAGCAGAGCCAGCTCAGCTCTGAGCCATTCACACATCTTCACCCTCAGTTTCCT CACTTGAGGAGTGGGATGGGGAGAACAGAGAGTAGCTGTGTTTGAATCCCTGTAGGAAAT GGTGAAGCATAGCTCTGGGTCTCCTGGGGGAGACCAGGCTTGGCTGCGGGAGAGCTGGCT GTTGCTGGACTACATGCTGGCCACTGCTGTGACCACGACACTGCTGGGGCAGCTTCTTCCA CAGTGATGCCTACTGATGCTTCAGTGCCTCTGCACACCGCCCATTCCACTTCCTCCTTCCCC ACAGGGCAGGTGGGGAAGCAGTTTGGCCCAGCCCAAGGAGACCCCACCTTGAGCCTTATT
TCCTAATGGGTCCACCTCTCATCTGCATCTTTCACACCTCCCAGCTTCTGCCCAACCTTCAG
CAGTGACAAGTCCCCAAGAGACTCGCCTGAGCAGCTTGGGCTGCTTTTCATTTCCACCTGT
CAGGATGCCTGTGGTCATGCTCTCAGCTCCACCTGGCATGAGAAGGGATCCTGGCCTCTGG
CATATTCATCAAGTATGAGTTCTGGGGATGAGTCACTGTAATGATGTGAGCAGGGAGCCTT
CCTCCCTGGGCCACCTGCAGAGAGCTTTCCCACCAACTTTGTACCTTGATTGCCTTACAAA
GTTATTTGTTTACAAACAGCGACCATATAAAAGCCTCCTGCCCCAAAGCTTGTGGGCACAT
GGGCACATACAGACTCACATACAGACACACACATATATGTACAGACATGTACTCTCACAC
AAAGTCCCATCACTGAGGGAGCCTAACCATGTCCCTGAACAAAAATTGGGCACTCATCTAT TCCTTTTCTCTTGTGTCCCTACTCATTGAAACCAAACTCTGGAAAGGACCCAATGTACCAGT ATTTATACCTCTAATGAAGCACAGAGAGAGGAAGAGAGCTGCTTAAACTCACACAACAAT GAACTGCAGACACAGCTGTTCTCTCCCTCTCTCCTTCCCAGAGCAATTTATACTTTACCCTC AGGCTGTCCTCTGGGGAGAAGGTGCCATGGTCTTAGGTGTCTGTGCCCCAGGACAGACCCT AGGACCCTAAATCCAATAGAAAATGCATATCTTTGCTCCACTTTCAGCCAGGCTGGAGCAA GGTACCTTTTCTTAGGATCTTGGGAGGGAATGGATGCCCCTCTCTGCATGATCTTGTTGAG GCATTTAGCTGCCATGCACCTGTCCCCCTTTAATACTGGGCATTTTAAAGCCATCTCAAGA GGCATCTTCTACATGTTTTGTACGCATTAAAATAATTTCAAAGATATCTGAGAAAAGCCGA TATTTGCCATTCTTCCTATATCCTGGAATATATCTTGCATCCTGAGTTTATAATAATAAATA ATATTCTACCTTGGAAAAAAAAAAAAAAA (SEQ ID NO: 57,997)
[00161] The MYD88 (isoform 2) nucleic acid sequence is:
[00162] AGATTCCTACTTCTTACGCCCCCCACATCACCCGCCTCGAGACCTCAAGGGTAGAGGTGGG CACCCCCGCCTCCGCACTTTTGCTCGGGGCTCCAGATTGTAGGGCAGGGCGGCGCTTCTCG GAAAGCGAAAGCCGGCGGGGCGGGGCGGGTGCCGCAGGAGAAAGAGGAAGCGCTGGCA GACAATGCGACCCGACCGCGCTGAGGCTCCAGGACCGCCCGCCATGGCTGCAGGAGGTCC CGGCGCGGGGTCTGCGGCCCCGGTCTCCTCCACATCCTCCCTTCCCCTGGCTGCTCTCAAC ATGCGAGTGCGGCGCCGCCTGTCTCTGTTCTTGAACGTGCGGACACAGGTGGCGGCCGACT GGACCGCGCTGGCGGAGGAGATGGACTTTGAGTACTTGGAGATCCGGCAACTGGAGACAC AAGCGGACCCCACTGGCAGGCTGCTGGACGCCTGGCAGGGACGCCCTGGCGCCTCTGTAG GCCGACTGCTCGAGCTGCTTACCAAGCTGGGCCGCGACGACGTGCTGCTGGAGCTGGGAC CCAGCATTGAGGAGGATTGCCAAAAGTATATCTTGAAGCAGCAGCAGGAGGAGGCTGAGA AGCCTTTACAGGTGGCCGCTGTAGACAGCAGTGTCCCACGGACAGCAGAGCTGGCGGGCA TCACCACACTTGATGACCCCCTGGGGCATATGCCTGAGCGTTTCGATGCCTTCATCTGCTAT TGCCCCAGCGACATCCAGTTTGTGCAGGAGATGATCCGGCAACTGGAACAGACAAACTAT CGACTGAAGTTGTGTGTGTCTGACCGCGATGTCCTGCCTGGCACCTGTGTCTGGTCTATTGC TAGTGAGCTCATCGAAAAGAGGTGCCGCCGGATGGTGGTGGTTGTCTCTGATGATTACCTG CAGAGCAAGGAATGTGACTTCCAGACCAAATTTGCACTCAGCCTCTCTCCAGGTGCCCATC AGAAGCGACTGATCCCCATCAAGTACAAGGCAATGAAGAAAGAGTTCCCCAGCATCCTGA GGTTCATCACTGTCTGCGACTACACCAACCCCTGCACCAAATCTTGGTTCTGGACTCGCCTT GCCAAGGCCTTGTCCCTGCCCTGAAGACTGTTCTGAGGCCCTGGGTGTGTGTGTATCTGTC TGCCTGTCCATGTACTTCTGCCCTGCCTCCTCCTTTCGTTGTAGGAGGAATCTGTGCTCTAC TTACCTCTCAATTCCTGGAGATGCCAACTTCACAGACACGTCTGCAGCAGCTGGACATCAC ATTTCATGTCCTGCATGGAACCAGTGGCTGTGAGTGGCATGTCCACTTGCTGGATTATCAG CCAGGACACTATAGAACAGGACCAGCTGAGACTAAGAAGGACCAGCAGAGCCAGCTCAG CTCTGAGCCATTCACACATCTTCACCCTCAGTTTCCTCACTTGAGGAGTGGGATGGGGAGA ACAGAGAGTAGCTGTGTTTGAATCCCTGTAGGAAATGGTGAAGCATAGCTCTGGGTCTCCT GGGGGAGACCAGGCTTGGCTGCGGGAGAGCTGGCTGTTGCTGGACTACATGCTGGCCACT GCTGTGACCACGACACTGCTGGGGCAGCTTCTTCCACAGTGATGCCTACTGATGCTTCAGT GCCTCTGCACACCGCCCATTCCACTTCCTCCTTCCCCACAGGGCAGGTGGGGAAGCAGTTT GGCCCAGCCCAAGGAGACCCCACCTTGAGCCTTATTTCCTAATGGGTCCACCTCTCATCTG CATCTTTCACACCTCCCAGCTTCTGCCCAACCTTCAGCAGTGACAAGTCCCCAAGAGACTC GCCTGAGCAGCTTGGGCTGCTTTTCATTTCCACCTGTCAGGATGCCTGTGGTCATGCTCTCA GCTCCACCTGGCATGAGAAGGGATCCTGGCCTCTGGCATATTCATCAAGTATGAGTTCTGG GGATGAGTCACTGTAATGATGTGAGCAGGGAGCCTTCCTCCCTGGGCCACCTGCAGAGAG CTTTCCCACCAACTTTGTACCTTGATTGCCTTACAAAGTTATTTGTTTACAAACAGCGACCA TATAAAAGCCTCCTGCCCCAAAGCTTGTGGGCACATGGGCACATACAGACTCACATACAG ACACACACATATATGTACAGACATGTACTCTCACACACACAGGCACCAGCATACACACGT TTTTCTAGGTACAGCTCCCAGGAACAGCTAGGTGGGAAAGTCCCATCACTGAGGGAGCCT AACCATGTCCCTGAACAAAAATTGGGCACTCATCTATTCCTTTTCTCTTGTGTCCCTACTCA TTGAAACCAAACTCTGGAAAGGACCCAATGTACCAGTATTTATACCTCTAATGAAGCACA GAGAGAGGAAGAGAGCTGCTTAAACTCACACAACAATGAACTGCAGACACAGCTGTTCTC TCCCTCTCTCCTTCCCAGAGCAATTTATACTTTACCCTCAGGCTGTCCTCTGGGGAGAAGGT GCCATGGTCTTAGGTGTCTGTGCCCCAGGACAGACCCTAGGACCCTAAATCCAATAGAAA ATGCATATCTTTGCTCCACTTTCAGCCAGGCTGGAGCAAGGTACCTTTTCTTAGGATCTTGG GAGGGAATGGATGCCCCTCTCTGCATGATCTTGTTGAGGCATTTAGCTGCCATGCACCTGT CCCCCTTTAATACTGGGCATTTTAAAGCCATCTCAAGAGGCATCTTCTACATGTTTTGTACG CATTAAAATAATTTCAAAGATATCTGAGAAAAGCCGATATTTGCCATTCTTCCTATATCCT GGAATATATCTTGCATCCTGAGTTTATAATAATAAATAATATTCTACCTTGGAAAAAAAAA AAAAAA (SEQ ID NO: 57,998)
[00163] The MYD88 (isoform 3) nucleic acid sequence is:
[00164] AGATTCCTACTTCTTACGCCCCCCACATCACCCGCCTCGAGACCTCAAGGGTAGAGGTGGG CACCCCCGCCTCCGCACTTTTGCTCGGGGCTCCAGATTGTAGGGCAGGGCGGCGCTTCTCG GAAAGCGAAAGCCGGCGGGGCGGGGCGGGTGCCGCAGGAGAAAGAGGAAGCGCTGGCA GACAATGCGACCCGACCGCGCTGAGGCTCCAGGACCGCCCGCCATGGCTGCAGGAGGTCC CGGCGCGGGGTCTGCGGCCCCGGTCTCCTCCACATCCTCCCTTCCCCTGGCTGCTCTCAAC
ATGCGAGTGCGGCGCCGCCTGTCTCTGTTCTTGAACGTGCGGACACAGGTGGCGGCCGACT
GGACCGCGCTGGCGGAGGAGATGGACTTTGAGTACTTGGAGATCCGGCAACTGGAGACAC
AAGCGGACCCCACTGGCAGGCTGCTGGACGCCTGGCAGGGACGCCCTGGCGCCTCTGTAG
GCCGACTGCTCGAGCTGCTTACCAAGCTGGGCCGCGACGACGTGCTGCTGGAGCTGGGAC
CCAGCATTGGGCATATGCCTGAGCGTTTCGATGCCTTCATCTGCTATTGCCCCAGCGACAT
CCAGTTTGTGCAGGAGATGATCCGGCAACTGGAACAGACAAACTATCGACTGAAGTTGTG
TGTGTCTGACCGCGATGTCCTGCCTGGCACCTGTGTCTGGTCTATTGCTAGTGAGCTCATCG
AAAAGAGGTGCCGCCGGATGGTGGTGGTTGTCTCTGATGATTACCTGCAGAGCAAGGAAT
GTGACTTCCAGACCAAATTTGCACTCAGCCTCTCTCCAGGTGCCCATCAGAAGCGACTGAT
CCCCATCAAGTACAAGGCAATGAAGAAAGAGTTCCCCAGCATCCTGAGGTTCATCACTGT
CTGCGACTACACCAACCCCTGCACCAAATCTTGGTTCTGGACTCGCCTTGCCAAGGCCTTG
TCCCTGCCCTGAAGACTGTTCTGAGGCCCTGGGTGTGTGTGTATCTGTCTGCCTGTCCATGT
ACTTCTGCCCTGCCTCCTCCTTTCGTTGTAGGAGGAATCTGTGCTCTACTTACCTCTCAATT
CCTGGAGATGCCAACTTCACAGACACGTCTGCAGCAGCTGGACATCACATTTCATGTCCTG
CATGGAACCAGTGGCTGTGAGTGGCATGTCCACTTGCTGGATTATCAGCCAGGACACTATA
GAACAGGACCAGCTGAGACTAAGAAGGACCAGCAGAGCCAGCTCAGCTCTGAGCCATTCA
CACATCTTCACCCTCAGTTTCCTCACTTGAGGAGTGGGATGGGGAGAACAGAGAGTAGCT
GTGTTTGAATCCCTGTAGGAAATGGTGAAGCATAGCTCTGGGTCTCCTGGGGGAGACCAG
GCTTGGCTGCGGGAGAGCTGGCTGTTGCTGGACTACATGCTGGCCACTGCTGTGACCACGA
CACTGCTGGGGCAGCTTCTTCCACAGTGATGCCTACTGATGCTTCAGTGCCTCTGCACACC
GCCCATTCCACTTCCTCCTTCCCCACAGGGCAGGTGGGGAAGCAGTTTGGCCCAGCCCAAG
GAGACCCCACCTTGAGCCTTATTTCCTAATGGGTCCACCTCTCATCTGCATCTTTCACACCT
CCCAGCTTCTGCCCAACCTTCAGCAGTGACAAGTCCCCAAGAGACTCGCCTGAGCAGCTTG
GGCTGCTTTTCATTTCCACCTGTCAGGATGCCTGTGGTCATGCTCTCAGCTCCACCTGGCAT
GAGAAGGGATCCTGGCCTCTGGCATATTCATCAAGTATGAGTTCTGGGGATGAGTCACTGT
AATGATGTGAGCAGGGAGCCTTCCTCCCTGGGCCACCTGCAGAGAGCTTTCCCACCAACTT
TGTACCTTGATTGCCTTACAAAGTTATTTGTTTACAAACAGCGACCATATAAAAGCCTCCT
GCCCCAAAGCTTGTGGGCACATGGGCACATACAGACTCACATACAGACACACACATATAT
GTACAGACATGTACTCTCACACACACAGGCACCAGCATACACACGTTTTTCTAGGTACAGC
TCCCAGGAACAGCTAGGTGGGAAAGTCCCATCACTGAGGGAGCCTAACCATGTCCCTGAA
CAAAAATTGGGCACTCATCTATTCCTTTTCTCTTGTGTCCCTACTCATTGAAACCAAACTCT
GGAAAGGACCCAATGTACCAGTATTTATACCTCTAATGAAGCACAGAGAGAGGAAGAGAG
CTGCTTAAACTCACACAACAATGAACTGCAGACACAGCTGTTCTCTCCCTCTCTCCTTCCCA
GAGCAATTTATACTTTACCCTCAGGCTGTCCTCTGGGGAGAAGGTGCCATGGTCTTAGGTG
TCTGTGCCCCAGGACAGACCCTAGGACCCTAAATCCAATAGAAAATGCATATCTTTGCTCC
ACTTTCAGCCAGGCTGGAGCAAGGTACCTTTTCTTAGGATCTTGGGAGGGAATGGATGCCC CTCTCTGCATGATCTTGTTGAGGCATTTAGCTGCCATGCACCTGTCCCCCTTTAATACTGGG CATTTTAAAGCCATCTCAAGAGGCATCTTCTACATGTTTTGTACGCATTAAAATAATTTCAA AGATATCTGAGAAAAGCCGATATTTGCCATTCTTCCTATATCCTGGAATATATCTTGCATCC TGAGTTTATAATAATAAATAATATTCTACCTTGGAAAAAAAAAAAAAAA (SEQ ID NO: 57,999)
[00165] The MYD88 (isoform 4) nucleic acid sequence is:
[00166] AGATTCCTACTTCTTACGCCCCCCACATCACCCGCCTCGAGACCTCAAGGGTAGAGGTGGG CACCCCCGCCTCCGCACTTTTGCTCGGGGCTCCAGATTGTAGGGCAGGGCGGCGCTTCTCG GAAAGCGAAAGCCGGCGGGGCGGGGCGGGTGCCGCAGGAGAAAGAGGAAGCGCTGGCA GACAATGCGACCCGACCGCGCTGAGGCTCCAGGACCGCCCGCCATGGCTGCAGGAGGTCC CGGCGCGGGGTCTGCGGCCCCGGTCTCCTCCACATCCTCCCTTCCCCTGGCTGCTCTCAAC ATGCGAGTGCGGCGCCGCCTGTCTCTGTTCTTGAACGTGCGGACACAGGTGGCGGCCGACT GGACCGCGCTGGCGGAGGAGATGGACTTTGAGTACTTGGAGATCCGGCAACTGGAGACAC AAGCGGACCCCACTGGCAGGCTGCTGGACGCCTGGCAGGGACGCCCTGGCGCCTCTGTAG GCCGACTGCTCGAGCTGCTTACCAAGCTGGGCCGCGACGACGTGCTGCTGGAGCTGGGAC CCAGCATTGAGGAGGATTGCCAAAAGTATATCTTGAAGCAGCAGCAGGAGGAGGCTGAGA AGCCTTTACAGGTGGCCGCTGTAGACAGCAGTGTCCCACGGACAGCAGAGCTGGCGGGCA TCACCACACTTGATGACCCCCTGGGTGCCGCCGGATGGTGGTGGTTGTCTCTGATGATTAC CTGCAGAGCAAGGAATGTGACTTCCAGACCAAATTTGCACTCAGCCTCTCTCCAGGTGCCC ATCAGAAGCGACTGATCCCCATCAAGTACAAGGCAATGAAGAAAGAGTTCCCCAGCATCC TGAGGTTCATCACTGTCTGCGACTACACCAACCCCTGCACCAAATCTTGGTTCTGGACTCG CCTTGCCAAGGCCTTGTCCCTGCCCTGAAGACTGTTCTGAGGCCCTGGGTGTGTGTGTATCT GTCTGCCTGTCCATGTACTTCTGCCCTGCCTCCTCCTTTCGTTGTAGGAGGAATCTGTGCTC TACTTACCTCTCAATTCCTGGAGATGCCAACTTCACAGACACGTCTGCAGCAGCTGGACAT CACATTTCATGTCCTGCATGGAACCAGTGGCTGTGAGTGGCATGTCCACTTGCTGGATTAT CAGCCAGGACACTATAGAACAGGACCAGCTGAGACTAAGAAGGACCAGCAGAGCCAGCT CAGCTCTGAGCCATTCACACATCTTCACCCTCAGTTTCCTCACTTGAGGAGTGGGATGGGG AGAACAGAGAGTAGCTGTGTTTGAATCCCTGTAGGAAATGGTGAAGCATAGCTCTGGGTC TCCTGGGGGAGACCAGGCTTGGCTGCGGGAGAGCTGGCTGTTGCTGGACTACATGCTGGC CACTGCTGTGACCACGACACTGCTGGGGCAGCTTCTTCCACAGTGATGCCTACTGATGCTT CAGTGCCTCTGCACACCGCCCATTCCACTTCCTCCTTCCCCACAGGGCAGGTGGGGAAGCA GTTTGGCCCAGCCCAAGGAGACCCCACCTTGAGCCTTATTTCCTAATGGGTCCACCTCTCA TCTGCATCTTTCACACCTCCCAGCTTCTGCCCAACCTTCAGCAGTGACAAGTCCCCAAGAG ACTCGCCTGAGCAGCTTGGGCTGCTTTTCATTTCCACCTGTCAGGATGCCTGTGGTCATGCT CTCAGCTCCACCTGGCATGAGAAGGGATCCTGGCCTCTGGCATATTCATCAAGTATGAGTT CTGGGGATGAGTCACTGTAATGATGTGAGCAGGGAGCCTTCCTCCCTGGGCCACCTGCAG AGAGCTTTCCCACCAACTTTGTACCTTGATTGCCTTACAAAGTTATTTGTTTACAAACAGCG ACCATATAAAAGCCTCCTGCCCCAAAGCTTGTGGGCACATGGGCACATACAGACTCACAT ACAGACACACACATATATGTACAGACATGTACTCTCACACACACAGGCACCAGCATACAC ACGTTTTTCTAGGTACAGCTCCCAGGAACAGCTAGGTGGGAAAGTCCCATCACTGAGGGA GCCTAACCATGTCCCTGAACAAAAATTGGGCACTCATCTATTCCTTTTCTCTTGTGTCCCTA CTCATTGAAACCAAACTCTGGAAAGGACCCAATGTACCAGTATTTATACCTCTAATGAAGC ACAGAGAGAGGAAGAGAGCTGCTTAAACTCACACAACAATGAACTGCAGACACAGCTGTT CTCTCCCTCTCTCCTTCCCAGAGCAATTTATACTTTACCCTCAGGCTGTCCTCTGGGGAGAA GGTGCCATGGTCTTAGGTGTCTGTGCCCCAGGACAGACCCTAGGACCCTAAATCCAATAGA AAATGCATATCTTTGCTCCACTTTCAGCCAGGCTGGAGCAAGGTACCTTTTCTTAGGATCTT GGGAGGGAATGGATGCCCCTCTCTGCATGATCTTGTTGAGGCATTTAGCTGCCATGCACCT GTCCCCCTTTAATACTGGGCATTTTAAAGCCATCTCAAGAGGCATCTTCTACATGTTTTGTA CGCATTAAAATAATTTCAAAGATATCTGAGAAAAGCCGATATTTGCCATTCTTCCTATATC CTGGAATATATCTTGCATCCTGAGTTTATAATAATAAATAATATTCTACCTTGGAAAAAAA AAAAAAAA (SEQ ID NO: 58,000)
[00167] The MYD88 (isoform 5) nucleic acid sequence is:
[00168] AGATTCCTACTTCTTACGCCCCCCACATCACCCGCCTCGAGACCTCAAGGGTAGAGGTGGG CACCCCCGCCTCCGCACTTTTGCTCGGGGCTCCAGATTGTAGGGCAGGGCGGCGCTTCTCG GAAAGCGAAAGCCGGCGGGGCGGGGCGGGTGCCGCAGGAGAAAGAGGAAGCGCTGGCA GACAATGCGACCCGACCGCGCTGAGGCTCCAGGACCGCCCGCCATGGCTGCAGGAGGTCC CGGCGCGGGGTCTGCGGCCCCGGTCTCCTCCACATCCTCCCTTCCCCTGGCTGCTCTCAAC ATGCGAGTGCGGCGCCGCCTGTCTCTGTTCTTGAACGTGCGGACACAGGTGGCGGCCGACT GGACCGCGCTGGCGGAGGAGATGGACTTTGAGTACTTGGAGATCCGGCAACTGGAGACAC AAGCGGACCCCACTGGCAGGCTGCTGGACGCCTGGCAGGGACGCCCTGGCGCCTCTGTAG GCCGACTGCTCGAGCTGCTTACCAAGCTGGGCCGCGACGACGTGCTGCTGGAGCTGGGAC CCAGCATTGGTGCCGCCGGATGGTGGTGGTTGTCTCTGATGATTACCTGCAGAGCAAGGAA TGTGACTTCCAGACCAAATTTGCACTCAGCCTCTCTCCAGGTGCCCATCAGAAGCGACTGA TCCCCATCAAGTACAAGGCAATGAAGAAAGAGTTCCCCAGCATCCTGAGGTTCATCACTGT CTGCGACTACACCAACCCCTGCACCAAATCTTGGTTCTGGACTCGCCTTGCCAAGGCCTTG TCCCTGCCCTGAAGACTGTTCTGAGGCCCTGGGTGTGTGTGTATCTGTCTGCCTGTCCATGT ACTTCTGCCCTGCCTCCTCCTTTCGTTGTAGGAGGAATCTGTGCTCTACTTACCTCTCAATT CCTGGAGATGCCAACTTCACAGACACGTCTGCAGCAGCTGGACATCACATTTCATGTCCTG CATGGAACCAGTGGCTGTGAGTGGCATGTCCACTTGCTGGATTATCAGCCAGGACACTATA GAACAGGACCAGCTGAGACTAAGAAGGACCAGCAGAGCCAGCTCAGCTCTGAGCCATTCA CACATCTTCACCCTCAGTTTCCTCACTTGAGGAGTGGGATGGGGAGAACAGAGAGTAGCT GTGTTTGAATCCCTGTAGGAAATGGTGAAGCATAGCTCTGGGTCTCCTGGGGGAGACCAG GCTTGGCTGCGGGAGAGCTGGCTGTTGCTGGACTACATGCTGGCCACTGCTGTGACCACGA CACTGCTGGGGCAGCTTCTTCCACAGTGATGCCTACTGATGCTTCAGTGCCTCTGCACACC GCCCATTCCACTTCCTCCTTCCCCACAGGGCAGGTGGGGAAGCAGTTTGGCCCAGCCCAAG
GAGACCCCACCTTGAGCCTTATTTCCTAATGGGTCCACCTCTCATCTGCATCTTTCACACCT
CCCAGCTTCTGCCCAACCTTCAGCAGTGACAAGTCCCCAAGAGACTCGCCTGAGCAGCTTG
GGCTGCTTTTCATTTCCACCTGTCAGGATGCCTGTGGTCATGCTCTCAGCTCCACCTGGCAT
GAGAAGGGATCCTGGCCTCTGGCATATTCATCAAGTATGAGTTCTGGGGATGAGTCACTGT
AATGATGTGAGCAGGGAGCCTTCCTCCCTGGGCCACCTGCAGAGAGCTTTCCCACCAACTT
TGTACCTTGATTGCCTTACAAAGTTATTTGTTTACAAACAGCGACCATATAAAAGCCTCCT
GCCCCAAAGCTTGTGGGCACATGGGCACATACAGACTCACATACAGACACACACATATAT
GTACAGACATGTACTCTCACACACACAGGCACCAGCATACACACGTTTTTCTAGGTACAGC
TCCCAGGAACAGCTAGGTGGGAAAGTCCCATCACTGAGGGAGCCTAACCATGTCCCTGAA
CAAAAATTGGGCACTCATCTATTCCTTTTCTCTTGTGTCCCTACTCATTGAAACCAAACTCT
GGAAAGGACCCAATGTACCAGTATTTATACCTCTAATGAAGCACAGAGAGAGGAAGAGAG
CTGCTTAAACTCACACAACAATGAACTGCAGACACAGCTGTTCTCTCCCTCTCTCCTTCCCA
GAGCAATTTATACTTTACCCTCAGGCTGTCCTCTGGGGAGAAGGTGCCATGGTCTTAGGTG
TCTGTGCCCCAGGACAGACCCTAGGACCCTAAATCCAATAGAAAATGCATATCTTTGCTCC
ACTTTCAGCCAGGCTGGAGCAAGGTACCTTTTCTTAGGATCTTGGGAGGGAATGGATGCCC
CTCTCTGCATGATCTTGTTGAGGCATTTAGCTGCCATGCACCTGTCCCCCTTTAATACTGGG
CATTTTAAAGCCATCTCAAGAGGCATCTTCTACATGTTTTGTACGCATTAAAATAATTTCAA
AGATATCTGAGAAAAGCCGATATTTGCCATTCTTCCTATATCCTGGAATATATCTTGCATCC
TGAGTTTATAATAATAAATAATATTCTACCTTGGAAAAAAAAAAAAAAA (SEQ ID NO:
58,001)
[00169] "HLA-DR" or "major histocompatibility complex, class II, DR" encompasses HLA-DRA (major
histocompatibility complex, class II, DR alpha) as well as HLA-DRB (major histocompatibility complex, class II, DR beta, e.g., beta 1-5). The accession numbers for protein and nucleic acid sequences for HLA-DRA include: NP 061984.2 and NM 019111.4. The 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 acid samples compositions and methods of isolation
[00170] 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.
[00171] 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.
[00172] 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. Examples of 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).
[00173] 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.
[00174] 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). 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.
[00175] 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.
NUCLEIC ACID PROBES
[00176] The present disclosure provides an array of nucleic acid probes. In certain embodiments, a probe may be present on a surface of a planar support, e.g., in the form of an array. Thus, the present disclosure provides a microchip having immobilized thereon a subject nucleic acid probe array. To be specific, an array of surface-bound polynucleotides (probes), as is commonly known in the art and described below, is not a mixture of surface-bound polynucleotides because the species of surface-bound (immobilized) polynucleotides are spatially distinct and the array is addressable.
[00177] A subject array comprises 1, 10, 100, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, or more probes. For example, a subject array can comprise from about 10 to about 10 , from about 10 to about 10 , from about 10 to about 10 , from about 10 to about 105 different nucleic acid probes.
[00178] In some embodiments, a subject array comprises from 5 to 10, from 10 to 50, from 50 to 100, from 102 to 2 x 102, from 2 x 102 to 5 x 102, from 5 x 102 to 103, from 103 to 5 x 103, from 5 x 103 to 104, from 5 x 104 to the complete set of probes having nucleotide sequences set forth in SEQ ID NOs:3-57,956.
[00179] A subject array can include nucleic acid probes that hybridize to a Borrelia spp. nucleic acid. For example, a subject array can include nucleic acid probes comprising nucleotide sequences set forth in SEQ ID NOs: 10, 12, 13, 15, etc. In addition to nucleic acid probes that hybridize to nucleic acid from Borrelia, 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.
[00180] 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. In some embodiments, a probe in a subject array has a length of from about 50 nt to about 100 nt, e.g., about 70 nt.
Detection methods
Real time PCR
[00181] Real time polymerase chain reaction (RT-PCR, also referred to as quantitative -PCR (QPCR)) 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
complementary DNA. In the first method, a DNA-binding dye can bind to all double-stranded (ds)DNA in PCR, resulting in fluorescence of the dye. 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
concentrations to be quantified. 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). With reference to a standard dilution, 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. To ensure accuracy in the quantification, 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. 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) can break the reporter-quencher proximity and thus can allow unquenched emission of fluorescence, which can be detected. 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. As the reaction commences, during the annealing stage of the PCR 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. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. Since the quantity of DNA doubles every cycle during the exponential phase, relative amounts of DNA can be calculated, (e.g. a sample with a Ct of 3 cycles earlier than another has 23 = 8 times more template). Amounts of nucleic acid (e.g., RNA or DNA) can be determined by comparing the results to a standard curve produced by a real-time PCR of serial dilutions (e.g. undiluted, 1 :4, 1 : 16, 1 :64) of a known amount of nucleic acid. 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). 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. Other examples of 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). When the probe is intact, fluorescent energy transfer can occur and the reporter dye fluorescent emission can be absorbed by the quenching dye. During the extension phase of the PCR cycle, 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;
incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment. The quantification may or may not include an amplification step. The quantitation may not be experimental.
Microchips
[00183] 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.
[00184] A microchip can comprise a substrate. Substrates can include, but are not limited to, glass and
modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, and plastics.
[00185] 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. For example, a subject microcip can comprise from about 10 to about 102, from about 102 to about 103, from about 103 to about 104, from about 104 to about 105 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.
[00186] In some embodiments, a subject microchip comprises from 5 to 10, from 10 to 50, from 50 to 100, from 102 to 2 x 102, from 2 x 102 to 5 x 102, from 5 x 102 to 103, from 103 to 5 x 103, from 5 x 103 to 104, from 5 x 104 to the complete set of probes having nucleotide sequences set forth in SEQ ID NOs:3-57,956. [00187] A subject microchip can include nucleic acid probes that hybridize to a Borrelia spp. nucleic acid. For example, a subject microchip can include nucleic acid probes comprising nucleotide sequences set forth in SEQ ID NOs: 10, 12, 13, 15, etc. In addition to nucleic acid probes that hybridize to nucleic acid from Borrelia, 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.
[00188] 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. In some embodiments, a probe in a subject array has a length of from about 50 nt to about 100 nt, e.g., about 70 nt.
[00189] In some embodiments, probes can comprise sequence information for a specific set of genes and/or species. In some embodiments, a microchip can comprise probes that are complementary to nucleic acid sequences in bacteria, protozoans, fungi, and/or viruses.
[00190] 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, Borrelia hispanica, Borrelia japonica, Borrelia latyschewii, Borrelia lonestari , Borrelia lusitaniae, Borrelia mazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi, Borrelia parked, Borrelia persica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, Borrelia vincentii). In some instances the tick-borne disease can originate from Borrelia burgdorferi.
[00191] 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). In some instances the tick-borne disease can originate from Anaplasma phagocytophilum/Erlichia.
[00192] 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 peacockii, Rickettsia prowazekii, Rickettsia hipicephaliRickettsia rickettsii, Rickettsia sibirica, Rickettsia slovaca, Rickettsia tamurae, Rickettsia typhi). [00193] 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 weissii), 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 queenslandensis, Bartonella quintana, Bartonella rattaustraliani, Bartonella rattimassiliensis, Bartonella rochalimae, Bartonella schoenbuchensis, Bartonella silvatica, Bartonella silvicola, Bartonella talpae, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii spp. arupensis, Bartonella vinsonii spp. berkhoffii, Bartonella vinsonii spp. vinsonii, Bartonella phoceensis, Bartonella washoensis, Candidatus Bartonella antechini, Candidatus Bartonella bandicootii, Candidatus Bartonella breitschwerdtii, Candidatus Bartonella durdenii, Candidatus Bartonella eldjazairii, Candidatus Bartonella mayotimonensis, Candidatus Bartonella melophagi, Candidatus Bartonella merieuxii, Candidatus Bartonella monaxi, Candidatus Bartonella rudakovii, Candidatus Bartonella tamiae, Candidatus Bartonella thailandensis, Candidatus Bartonella volans, Candidatus Bartonella woyliei).
[00194] 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).
[00195] 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 weilii, 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 genomospecies 5).
[00196] 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). In some instances, the tick-borne disease can originate from Babesia microti. In some instances, the tick-borne disease can originate from Babesia duncani.
[00197] A probe can comprise a sequence that can be complementary to a nucleic acid sequence in the genome Toxoplasma (e.g., Toxoplasma gondii). In some instances, the tick-borne disease can originate from Toxoplasma gondii.
[00198] 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 oleophila, Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candida rugosa, Candida sake, Candida shehatea, Candida temnochilae, Candida tenuis, Candida theae, Candida tropicalis, Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candida subhashii, Candida viswanathii, Candida utilis).
[00199] 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).
[00200] 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.)
[00201] 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.
[00202] 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.
[00203] 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,
extrachromasomal 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), can be treated with Rnase or base (e.g., NaOH) to hydrolyze the RNA. 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.
[00204] The test sample and the reference sample can be applied to a microchip to contact multiple spots
simultaneously. 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. During the incubation period, nucleic acids in the nucleic acid sample can be in intimate contact with the microchip probes.
[00205] 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. In addition to photomultiplier tubes, detectors can include cameras that use charge coupled device (CCD) and complementary metal oxide silicon (CMOS) chips.
[00206] The ratio of the intensities of the two dyes from the test sample and the reference sample can be
compared for each probe. 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. The M vs. A plot is also referred to as the R vs. I plot, where R is a ratio, such as R = log2(Red/Green) and I is an intensity, such as I = log VRed* Green . 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
[00207] Deep sequencing can use techniques in Next Generation Sequencing (NGS). 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.
[00208] 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.
[00209] 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. In some cases, 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
Biotechnologies(Madison, WI).
[00210] 5' phosphorylated ends of nucleic acids can be ligated to an adapter. 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.
[00211] In some embodiments, a single-stranded adapter can comprises two or more sequences that can
hybridize with one another. When two such hybridizable sequences are contained in a single-stranded adapter, hybridization can yield a hairpin structure (hairpin adapter). When two hybridized regions of an adapter are separated from one another by a non-hybridized region, a "bubble" structure can result.
[00212] 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.
[00213] Adapters can be manipulated prior to combining with target nucleic acids. For example, terminal
phosphates can be added or removed. 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. one or more nucleotides selected at random from a set of two or more different nucleotides at one or more positions, with each of the different nucleotides selected at one or more positions represented in a pool of adapters comprising the random sequence), and combinations thereof. [00214] 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 (e.g, a target nucleic acid and an adapter) 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. Multiple 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. Ligation can be between polynucleotides having hybridizable sequences, such as complementary overhangs. Ligation can also be between two blunt ends.
[00215] 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.
[00216] 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 SOLiD™ technology, Life Technology, Inc., Carlsbad, Calif), sequencing by synthesis using modified nucleotides (such as commercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., San Diego, Calif, HeliScope™ by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, Calif), sequencing by ion detection
technologies (Ion Torrent, Inc., South San Francisco, Calif), sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View, Calif), nanopore -based sequencing technologies (for example, as developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and other known highly parallelized sequencing methods.
[00217] A sequencing technique that can be used in the methods of the provided invention can include, for example, Helicos True Single Molecule Sequencing (tSMS). In the tSMS technique, 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.
[00218] Another example of a 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. The result can be multiple copies of clonally amplified DNA fragments on each bead. In the second step, 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.
[00219] Another example of a DNA sequencing technique that can be used in the methods of the provided invention is SOLiD technology (Applied Biosystems). In 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. Alternatively, 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. [00220] 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.
[00221] Another example of a sequencing technology that can be used in the methods of the provided invention is Illumina sequencing. 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. Multiple cycles of the solid-phase amplification followed by denaturation can create several million clusters of approximately 1 ,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore-labeled, reversibly terminating nucleotides can be used to perform sequential sequencing. After nucleotide incorporation, a laser can be used to excite the fluorophores, and an image can be captured and the identity of the first base can be recorded. The 3' terminators and fluorophores from each incorporated base can be removed and the incorporation, detection and identification steps can be repeated.
[00222] Another example of a sequencing technology that can be used in the methods of the provided invention can include the single molecule, real-time (SMRT) technology of Pacific Biosciences. In SMRT, 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). A 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). It can take several milliseconds to incorporate a nucleotide into a growing strand. During this time, 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.
[00223] Another example of a sequencing technique that can be used in the methods of the provided invention is nanopore sequencing. 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. As a target nucleic acid passes through a nanopore, each nucleotide on the target nucleic acid can obstruct the nanopore to a different degree. Thus, 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.
[00224] Another example of 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. In one example of the technique, 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. In another example, 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.
[00225] Another example of a sequencing technique that can be used in the methods of the provided invention can involve using an electron microscope. In one example of the technique, 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. To begin the reconstruction process, short reads may be stitched together bioinformatically, e.g., by finding overlaps and extending them.
Data Analysis methods
Pathogen detection analysis
[00226] 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.
[00227] 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. [00228] 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.
[00229] The Ultra-Rapid Pathogen Identification in Minutes (URPI Pipeline) 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). To determine genes of other pathogenic origin, the 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
[00230] 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)), can classify differentially expressed genes through comparison of the expected expression of genes by protein class compared to the nucleic acid sample expression. For example, a data analysis program can compare the gene expression changes in the levels of cytokines from an experimental sample and a control sample.
[00231] A nucleic acid sample can comprise nucleic acids from pathogens (e.g., bacteria, protozoa, fungi,
viruses) and host nucleic acids. 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.
COMPUTER-IMPLEMENTED METHODS, SYSTEMS AND DEVICE
[00232] 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.
[00233] For example, 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.
[00234] For example, the methods of the present disclosure can involve inputting information regarding
detection of 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.
[00235] 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).
[00236] The present disclosure provides systems for executing the program described above, which system
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,
i) Computer Systems
[00237] 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. In certain embodiments, 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.
[00238] 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.
[00239] 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.
[00240] In use, the processing system 100 is adapted to allow data or information to be stored in and/or
retrieved from, via wired or wireless communication means, at least one database 116. The interface 112 may allow wired and/or wireless communication between the processing unit 102 and peripheral components that may serve a specialized purpose. In general, 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.
[00241] 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.
[00242] 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). Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets, and the Internet. For instance, when used in a LAN networking environment, the computing system environment 100 is connected to the LAN through a network interface or adapter. When used in a WAN networking environment, 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. In a networked environment, program modules depicted relative to the computing system environment 100, or portions thereof, may be stored in a remote memory storage device.
[00243] 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.
[00244] Certain embodiments may be described with reference to acts and symbolic representations of
operations that are performed by one or more computing devices, such as the computing system environment 100 of Figure 4. As such, it will be understood that such acts and operations, which are at times referred to as being computer-executed, include the manipulation by the processor of the computer of electrical signals representing data in a structured form. This manipulation transforms the data or maintains them at locations in the memory system of the computer, which reconfigures or otherwise alters the operation of the computer in a manner understood by those skilled in the art. The data structures in which data is maintained are physical locations of the memory that have particular properties defined by the format of the data. However, while an embodiment is being described in the foregoing context, it is not meant to be limiting as those of skill in the art will appreciate that the acts and operations described hereinafter may also be implemented in hardware.
[00245] 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.
[00246] Embodiments may be described in a general context of computer-executable instructions, such as
program modules, being executed by a computer. Generally, 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. In a distributed computing environment, program modules may be located in both local and remote computer storage media including memory storage devices.
Computer program products
[00247] 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. As discussed above, 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.). [00248] Computer programs (also known as programs, software, software applications, applications, components, or code) 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. As used herein, the term "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.
[00249] It will be apparent from this description that aspects of the present invention may be embodied, at least in part, in software, hardware, firmware, or any combination thereof. Thus, the techniques described herein are not limited to any specific combination of hardware circuitry and/or software, or to any particular source for the instructions executed by a computer or other data processing system. Rather, these techniques may be carried out in a computer system or other data processing system in response to one or more 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.
[00250] In addition, 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. In alternative embodiments, 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.
EXAMPLES
[00251] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. 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
[00252] A high-density 57,954-probe microarray was developed and called the TickChip™ (UCSF), for
simultaneous multiplexed detection of all known tick-borne pathogens, including a variety of B.
burgdorferi strains, tick-borne co-pathogens, and viruses.
[00253] 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 :
[00254] TABLE 1
Spirochetes: all known Borrelia species and strains; Treponema spp., Leptospira spp
a. Other bacteria: Ehrlichia spp., Anaplasma spp., Rickettsia spp., Bartonella spp.,
tularemia (Francisella tularensis), Q fever (Coxiella burnetii), Mycoplasma spp.,
Pseudomonas aeruginosa, Brucella spp., Chlamydia spp.
Protozoa: Babesia (microti, duncani, others), Toxoplasma spp.
Viruses: Tickbornc encephalitis virus, Powassan Virus, dengue virus, other bloodborne viral
targets
Fungi: Candida spp., Cryplococcus spp., Aspergillus Jiimigalus
[00255] An overview of one embodiment of the TickChip assay is shown in Figure 20. According to this
embodiment, 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.
EXPERIMENT 1
[00256] Using serially spiked controls, the limits of detection of this assay for detection of 4 representative vector-borne pathogens (B. burgdorferi, B. microti, Anaplasma phagocytophilum, and dengue type 1 virus) were found to be <1 -10 genomic copies per milliliter, with sensitivity comparable or superior to PCR. Clinically diagnosed acute Lyme disease samples were tested by TickChip and PCR (3 independent assays). By TickChip, 4 Borrelia positives at very low titer (cycle threshold range from 33 - 39 by qPCR) in 29 blood samples (13.8%) from acute Lyme disease patients presenting with fever and erythema migrans were detected, vs. only 2 of 29 (6.9%) by PCR.
METHODS AND RESULT
TickChip Analysis
[00257] For TickChip detection of nonviral pathogens, positive control samples for validation were quantified serial dilutions of Borrelia burgdorferi culture, Anaplasma phagocytophilum culture, and Babesia microti cultured in hamster blood into spiked negative blood matrices (blood from anonymized patients at UCSF). Samples were homogenized using an Omni Bead raptor (Omni Intl. Kennesaw, GA) and 0.1 mm silica beads (MP Biomedical, Solon, OH) prior to total nucleic acid extraction on the EZ1 instrument (Qiagen, Carlsbad, CA). 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.
[00258] For detection of viruses on the TickChip, using dengue as a positive validation control, quantified plasma samples positive for dengue virus provided by the American Red Cross (Stramer and Dodd) were first spun down to pellet and remove any cellular debris. The supernatant was then passed through a 0.22 μιη filter and then nucleic acid extraction was performed using the Qiagen EZ1 robot and Virus 2.0 kit (Qiagen, Carlsbad, CA). Extracted samples were then randomly amplified to generate a cDNA library as previously described [1,2]
[00259] Amplified samples were then pooled together, labeled with Cy3 fluorescent dye, and measured on a spectrophotometer to ensure adequate dye incorporation and cDNA concentration. All samples passed this quality check (QC) and were hybridized overnight on the TickChip microarray in a hybridization oven at 65°C (Agilent).
[00260] TickChip microarrays were washed and scanned at 2 μιη 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.
[00261] The bacterial pathogens examined include Borrelia burgdorferi, Anaplasma phagocytophilum, and
Babesia microti. 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 105-10° 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).
Similarly, we have used random amplification followed by BloodChip microarray analysis to detect dengue and HIV in clinically infected samples down to 1-10 copies per mL of serum. Rigorous assessment of limits of sensitivity by probit analysis, testing of a panel of bloodborne pathogens, and streamlining of the assay protocol are ongoing. Specifically, we will test the sample on 50 known Babesia positive donor blood samples provided by the American Red Cross and directly test the sensitivity of the BloodChip vs. PCR and serology for identification of B. microti and 100 whole blood samples from patients presenting to outpatient clinic with a fever and a characteristic erythema migrans rash and diagnosed with acute Lyme disease.
[00262] 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. As can be seen, 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).
[00263] 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
EXPERIMENT 2
[00264] 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).
METHODS AND RESULT
TickChip Analysis
[00265] 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).
[00266] Whole blood samples were extracted using a combination of freeze-thaw, bead-beat and pre-DNase followed by robotic extraction using the Qiagen EZ1 instrument. Viruses were extracted similarly with the omission of freeze-thaw and bead beating. Nucleic acid extracts were then amplified to cDNA using RT-PCR for the 16S rRNA of bacteria, 18S rRNA of fungi/parasites, or randomly for viruses, with the amplification tested using both individual and multiplexed reactions. An additional spiked 16S rRNA primer was added to specifically enhance detection of Borrelia (Figure 22). Custom TickChip microarrays were synthesized by Agilent Technologies and microarrays processed for scanning using a 3 μιη Agilent DNA microarray scanner. The amplified cDNA was labeled using Cy3 fluorescent dye and hybridized to the microarray overnight. Following scanning of the microarray, analysis for pathogen identification was performed using hierarchical cluster analysis and Z-score oligonucleotide analysis. [00267] Analytic sensitivity for detection of Borrelia burgdorferi, Babesia microti, Anaplasma
phagocytophilum, and dengue virus is 1-10 copies per niL with spiked samples. Results are provided in Table 2.
[00268] TABLE 2:
Figure imgf000071_0001
[00269] 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.
[00270] 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° - 104) 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.
[00271] 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. Note that 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).
[00272] 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°— 104) or in previously quantified RBC lysates from blood donors screening positive for Babesia infection (C512, C514, and C542). Note that 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).
EXPERIMENT 3
[00273] A TickChip for increased discrimination between clinically significant species of Borrelia, such as Borrelia burgodorferi and Borrelia miyamotoi, was designed. In addition to the 16S ribosomal RNA gene detection probes, 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). In conjunction with 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. An additional 616 70mer probes were designed to detect all of the recently described tickborne bunyaviruses, several of which (e.g., Heartland virus, SFTS virus) have been shown to be pathogenic to humans and the cause of vector-borne febrile illness (Figures 25A-R). SAMPLES AND TESTING
[00274] Aliquots of stock cultured Borrelia (B. burgdorferi, B. miyamotoi, and B. andersonii), Anaplasma phagcytophilum, and Babesia microti are available for testing. Such aliquots are cultured in hamster blood for use in spiking experiments. In addition, clinical samples, whole blood and serum from healthy patients for use as negative controls and spike -in matrix, are provided by the clinical labs as UCSF. Candida albicans, Aspergillus fulmigatus, Pseudomona aeruginosa, Staphylococcus aureus,
Staphylococcus epidermidis and Lactobacillus have been cultured for evaluating probe specificity. Clinically positive nucleic acid extracts for Anaplasma phagocytophilum wad Rickettsia philippi 364D as well as 150 from other suspect viral and bacterial tickborne infections (n=150) have been prepared. In addition, 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. 150 available human samples for clinical validation, including Babesia positive samples and serum samples positive for the mosquito-borne dengue and West Nile viruses, have been confirmed positive by immunofluorescent antibody (IF A) and/or PCR assays. 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.
[00275] 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. In addition, 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. 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.
[00276] Probit analysis of 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.
INTEGRATED CLINICAL LABORATORY WORKFLOW
[00277] 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. 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.
EXAMPLE 2
[00278] Deep sequencing of randomly amplified samples from patients with acute Lyme disease was
performed. We were able to show in 3 of 4 samples detection of Borrelia sequences, with analysis of over 100 million reads in under 10 minutes using an automated analysis computational pipeline called SURPI (sequenced-based ultra-r-apid pathogen identification). By deep sequencing, host transcriptome profiling by whole-exome sequencing of blood samples from 20 acute and 9 PTLDS (Post-treatment Lyme disease syndrome) patients and matched controls at 3 serially collected timepoints (pre -treatment, 1 month post-treatment, and at 6 months follow-up) was also performed. Transcriptome analysis has revealed a candidate set of -20 diagnostic host response markers that may prove 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. These studies have revealed that a next-generation assay incorporating both pathogen and host response markers can potentially be used to greatly enhance diagnosis of acute / chronic Lyme disease and related illnesses.
METHODS AND RESULTS
Deep Sequencing Analysis
[00279] 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.
[00280] For host transcriptome profiling, well-established techniques RNASeq techniques were used [5] to make deep sequencing libraries from blood samples from 20 acute and 9 PTLDS patients and matched controls at 3 serially collected timeoints. Transcriptome analysis was carried out using the
Tophat/Cufflinks pipeline [6], and data interpretation of significantly expressed genes performed by analysis from Ingenuity Pathways, Inc. 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.
[00281] Collectively, these studies have revealed that a next-generation assay incorporating both pathogen and host response markers can be used to greatly enhance diagnosis of acute / chronic Lyme disease and related illnesses.
SURPI (Sequence-based ultra-rapid pathogen identification) Computational Pipeline
[00282] 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. Key features of this pipeline are speed, wherein analysis of 20 million 250-bp reads can be completed in under 10 minutes and accuracy comparable to commonly used BLASTn, BOWTIE, and BWA algorithms by ROC curve analysis (Naccache, et al, manuscript in preparation). By receiver operating characteristic (ROC) curve analysis, SNAP has comparable sensitivity and specificity and higher speed (> ~30-times faster) than commonly used mapping algorithms (e.g., Bowtie, BWA). Initial end-to-end benchmarking of this system on clinical samples generating up to 100 million reads have completed in less than 1 hour. A graphical user interface (GUI), customized reference databases, and a pathogen diagnostic scoring system for SURPI can be used in clinical and research laboratories by technicians and lab directories.
[00283] Figure 18 depicts a sample workflow for an NGS-based diagnostic assay. (Upper) By NGS, herpes simplex virus 1 (HSV-1) was detected in cerebrospinal fluid (CSF) from an elderly patient presenting to the hospital with an unknown encephalitis syndrome. 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. (Lower) Borrelia burgdorferi sequences were detected in <24 hours by NGS assay in a patient with unexplained fever and rash.
[00284] REFERENCES
1. Greninger AL, Chen EC, Sittler T, Scheinerman A, Roubinian N, et al. (2010) A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America. PLoS One 5: el3381.
2. Chiu CY, Rouskin S, Koshy A, Urisman A, Fischer K, et al. (2006) Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult. Clin Infect Dis 43: e71-76.
3. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome -wide expression patterns. Proc Natl Acad Sci U S A 95: 14863-14868.
4. Swei A, Russell BJ, Naccache SN, Kabre B, Veeraraghavan N, et al. (2013) The Genome Sequence of Lone Star Virus, a Highly Divergent Bunyavirus Found in the Amblyomma americanum Tick. PLoS One 8: e62083.
5. Pease J, Sooknanan R (2012) A rapid, directional RNA-seq library preparation workflow for Illumina[reg] sequencing. Nat Meth 9.
6. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, et al. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7: 562-578.
7. Berendzen J, Bruno WJ, Cohn JD, Hengartner NW, Kuske CR, et al. (2012) Rapid phylogenetic and functional classification of short genomic fragments with signature peptides. BMC Res Notes 5: 460.
8. Zaharia M, W.J. B, Curtis K, Fox A, Patterson D, et al. (2011) Faster and more accurate sequence alignment with SNAP. arXivorg 1111.5572.
[00285] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method of detecting a tick-bome disease in a biological sample, the method comprising:
(a) contacting a biological sample with an array of immobilized nucleic acid probes, wherein the nucleic acid probes hybridize to one or more nucleic acids present in a tick-borne disease-inducing microorganism selected from the group consisting of bacteria, protozoans, fungi and viruses;
(b) obtaining hybridization information from said contacted array; and
(c) relating, with the aid of a computer processor, 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%.
2. The method of claim 1, wherein the nucleic acid probes comprises nucleotide sequences set forth in one or more of SEQ ID NOs:3-57956.
3. The method of claim 1, further comprising sequencing at least a portion of the biological sample.
4. The method of claim 1, wherein one or more of said nucleic acid probes hybridizes with 16S and/or 18S ribosomal nucleic acid of the one or more tick-borne disease-inducing microorganisms.
5. The method of claim 1, wherein the nucleic acid probes comprises from 102 to 103, from 103 to 104, or from 104 to 5 x 104, nucleotide sequences set forth in one or more of SEQ ID NOs:3-57956.
6. The method of claim 1, wherein the tick-borne disease-inducing microorganism is a Borrelia species.
7. A microchip, comprising:
(a) a substrate; and
(b) 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 hybridize to one or more nucleic acids of tick-borne disease-inducing microorganisms comprising at least any three of bacteria, protozoans, fungi, and viruses.
8. The microchip of claim 7, wherein the bacteria include Borrelia burgdorferi, Anaplasma phagocytophilum/Ehrlichia, Rickettsia, Bartonella, Francisella or Leptospira.
9. The microchip of claim 7, wherein the protozoans include Babesia microti, Babesia duncani or Toxoplasma gondii.
10. The microchip of claim 7, wherein the fungi include Candida spp or Cryptococcus spp.
11. The microchip of claim 7, wherein the viruses include tick-borne encephalitis virus, dengue virus, or a virus from the Bunyaviridae family of virses.
12. The microchip of claim 7, wherein 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.
13. The microchip of claim 7, wherein the number of the one or more polynucleotide molecules is at least 60,000.
14. The microchip of claim 7, wherein the length of each of the one or more
polynucleotide molecules is greater than 70 bp.
15. The microchip of claim 7, wherein the length of each of the one or more
polynucleotide molecules is less than 70 bp.
16. The microchip of claim 7, wherein the tick-borne disease is Lyme Disease.
17. The microchip of claim 7, wherein said tick-borne disease-inducing microorganisms comprise bacteria, protozoans and fungi.
18. The microchip of claim 7, wherein said tick-borne disease-inducing microorganisms comprise bacteria, protozoans and viruses.
19. The microchip of claim 7, wherein said tick-borne disease-inducing microorganisms comprise bacteria, fungi and viruses.
20. The microchip of claim 7, wherein said tick-borne disease-inducing microorganisms comprise protozoans, fungi and viruses.
21. The microchip of claim 7, wherein said tick-borne disease-inducing microorganisms comprise bacteria, protozoans, fungi and viruses.
22. A microchip, comprising:
(a) a substrate; and
(b) 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 hybridize to one or more nucleic acids of 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, Francisella 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.
23. A method of detecting a tick-borne disease, comprising:
(a) performing pathogen detection analysis on a biological sample obtained from a subject, wherein said biological sample comprises one or more nucleic acids;
performing host response analysis on the one or more nucleic acids; and relating, with the aid of a computer processor, 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.
24. The method of claim 1, wherein the biological sample is a blood sample.
25. The method of claim 1, further comprising RNA extraction and DNA extraction from the biological sample.
26. The method of claim 1, further comprising RNA extraction from peripheral blood mononuclear cells (PBMC) found in the biological sample.
27. The method of claim 1, wherein the pathogen detection analysis and the host response analysis are done simultaneously.
28. The method of claim 1, wherein the pathogen detection analysis and the host response analysis are done sequentially.
29. The method of claim 1, wherein the pathogen detection analysis is performed by deep sequencing, real time polymerase chain reaction, microarray or using a microchip.
30. The method of claim 7, wherein the microchip comprises one or more
polynucleotides that hybridize to one or more tick-borne disease-inducing microorganisms.
31. The method of claim 7, wherein the microchip comprises one or more
polynucleotides that hybridize to one or more gene expressed in a host in response to infection with a tick-borne disease-causing microoganism.
32. The method of claim 9, wherein the one or more polynucleotide molecules comprise at least one of IFIT2, IFITM2, SOCSl, SOCS3, STATl, STAT2, IRFl, TLRl, TLR2, TLR3, TNF, HLA-DQ, HLA-DR, and MYD88.
33. The method of claim 7, wherein the deep sequencing comprises:
(a) obtaining sequence information of said one or more nucleic acid molecules;
(b) subtracting human genomic information from the sequence information obtained in (a); and
(c) 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.
34. The method of claim 7 or 11, wherein the deep sequencing is performed with Illumina HiSeq or MiSeq.
35. The method of claim 11, further comprising establishing a transcriptome of the biological sample.
36. The method of claim 1, wherein the host response analysis is performed by deep sequencing, real time PCR, microarray or using a microchip.
37. The method of claim 1, wherein the information obtained from the host response analysis comprises a differential expression level of one or more genes that are related to the tick- borne disease.
38. The method of claim 15, wherein the differential expression level of said one or more genes is measured for PBMC found in the biological sample.
39. The method of claim 1, wherein the pathogen detection analysis allows direct detection of all bacteria species of Borrelia.
40. The method of claim 1, wherein the pathogen detection analysis allows direct detection of all virus species of Bunyaviridae.
41. The method of claim 1, wherein the subject has acute or chronic tick-borne disease.
42. The method of claim 1, wherein no tick-borne related pathogen can be detected in the biological sample.
43. The method of claim 1, wherein the pathogen detection analysis is based on the sequence information of 16S and 18S ribosomal RNA of one or more pathogens capable of inducing the tick-borne disease.
44. The method of claim 1, wherein said detecting the tick-borne disease is at a sensitivity of at least 90% and at a specificity of at least 90%.
45. A method of detecting a tick-borne disease at a sensitivity of at least 90% and a specificity of at least 90%.
46. A method of detecting a tick-borne disease based on the sequence information of 16S and 18S ribosomal RNA 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%.
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