# US 2018/0148780 A1 · Next-Generation Sequencing-Based Genotyping Assay for Human Papilloma Virus (HPV) > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0148780 A1 - Document ID: US20180148780A1 - Status: Published application - Kind: A1 - Application number: 15/575,977 - Filed: 2016-05-20 - Published: 2018-05-31 - Earliest claimed: 2015-05-22 - Assignees: University of Maryland, Baltimore; Office of Technology Transfer, National Institutes of Health - Applicants: University of Maryland, Baltimore; Office of Technology Transfer, National Institutes of Health - Inventors: Kevin CULLEN; Nicholas AMBULOS; Jennifer TROYER - Web page: https://patentgrep.com/patent/US20180148780A1 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q 1/708: {for papilloma} - A61P 31/20: for DNA viruses - C12N 2710/20021: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12Q 2535/122: Massive parallel sequencing - G01N 2333/025: Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus - G01N 2800/26: Infectious diseases, e.g. generalised sepsis - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Disclosed herein are compositions and methods for detecting and diagnosing HPV in subjects. The method includes using Next Gen Sequencing for detecting HPV in a sample and determining the specific subtype of HPV infection based on the sequencing data. ## Claims ### Claim 1 A method for detecting HPV nucleic acid in a biological sample of a subject suspected of having an HPV infection or an HPV associated disease, the method comprising: (a) isolating a nucleic acid sample from a biological sample obtained from the subject; (b) determining the nucleic acid sequence of the nucleic acid sample using a next generation (Next Gen) sequencing assay; and (c) determining the genotype of HPV in the sample, wherein the method of determining the nucleic acid sequence of the nucleic acid sample comprises: (i) contacting the nucleic acid sample with at least one forward PCR primer, (ii) contacting the nucleic acid sample with at least one reverse PCR primer, (iii) amplifying the HPV nucleic acid using PCR, and (vi) sequencing the amplified nucleic acid products using a Next Gen Sequencer, and wherein the at least one forward PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and further comprises a nucleic acid sequence for use as a sequencing adaptor. ### Claim 2 (canceled) ### Claim 3 (canceled) ### Claim 4 (depends on 1) The method of claim 1, wherein the at least one forward PCR primer comprises at least two forward PCR primers, wherein each of the at least two forward PCR primers comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and further wherein each of the at least two forward PCR primers further comprises a nucleic acid sequence for use as a sequencing adaptor. ### Claim 5 (depends on 1) The method of claim 1, wherein the at least one forward PCR primer comprises at least nine forward PCR primers, wherein each of the at least nine forward PCR primers comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and further wherein each of the at least nine forward PCR primers further comprises a nucleic acid sequence for use as a sequencing adaptor. ### Claim 6 (depends on 1) The method of claim 1, wherein the sequencing adaptor comprises or consists of the sequence set forth in SEQ ID NO:10. ### Claim 7 (depends on 1) The method of claim 1, wherein the at least one reverse PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and wherein each of the at least one reverse PCR primers comprises a nucleic acid sequence for use as a barcode and a nucleic acid sequence for use as a sequencing adaptor. ### Claim 8 (depends on 1) The method of claim 1, wherein the at least one reverse PCR primer comprises at least two reverse PCR primers, wherein each of the at least two reverse PCR primers comprises a nucleic acid sequence selected from the group comprising SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, further wherein each of the at least two reverse PCR primers comprises a nucleic acid sequence for use as a barcode and a nucleic acid sequence for use as a sequencing adaptor. ### Claim 9 (depends on 1) The method of claim 1, wherein the at least one reverse PCR primer comprises at least three reverse PCR primers, wherein at least one of the reverse PCR primers comprises the nucleic acid sequence set forth in SEQ ID NO:11, at least one of the PCR primers comprises the nucleic acid sequence set forth in SEQ ID NO:12, and at least one of the PCR primers comprises the nucleic acid sequence set forth in SEQ ID NO:13, further wherein each of the at least one reverse PCR primers comprises a nucleic acid sequence for use as a barcode and a nucleic acid sequence for use as a sequencing adaptor. ### Claim 10 (depends on 7) The method of claim 7, wherein the sequencing adaptor comprises or consists of the sequence set forth in SEQ ID NO: 17. ### Claim 11 (canceled) ### Claim 12 (canceled) ### Claim 13 (canceled) ### Claim 14 (depends on 1) The method of claim 1, wherein the method of detecting HPV nucleic acid in the subject comprises aligning a set of sequence data reads to at least one HPV reference sequence, identifying sequencing reads that align to the at least one HPV reference sequence, evaluating the nucleic information of the identified sequencing reads to identify at least one HPV subtype-specific genetic variation, and determining the subtype of the HPV based on the at least one HPV subtype-specific genetic variations. ### Claim 15 (canceled) ### Claim 16 (depends on 14) The method of claim 14, wherein multiple HPV subtypes are determined for a sample from a subject. ### Claim 17 (canceled) ### Claim 18 (canceled) ### Claim 19 (depends on 1) The method of claim 1, wherein the method of detecting HPV nucleic acid in the subject comprises aligning a set of sequence data reads to at least one HPV reference sequence, identifying sequencing reads that align to the at least one HPV reference sequence, evaluating the nucleic information of the identified sequencing reads to identify at least one HPV genotype. ### Claim 20 (canceled) ### Claim 21 (depends on 19) The method of claim 19, wherein multiple HPV genotypes are determined for a sample from a subject. ### Claim 22 (depends on 19) The method of claim 19, wherein multiple HPV genotypes are determined when at least 1% of the sequencing reads for a sample carry a genotype specific nucleic acid. ### Claim 23 (canceled) ### Claim 24 (depends on 1) The method of claim 1, further comprising (f) diagnosing the subject with infection by HPV. ### Claim 25 (depends on 24) The method of claim 24, further comprising (g) providing a treatment to the subject on the basis of the diagnosis. ### Claim 26 (canceled) ### Claim 27 (canceled) ### Claim 28 (canceled) ### Claim 29 (canceled) ### Claim 30 A composition comprising at least one PCR primer, wherein the at least one PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and further comprises a nucleic acid sequence for use as a sequencing adaptor. ### Claim 31 (depends on 30) The composition of claim 30, wherein the nucleic acid sequence for use as a sequencing adaptor consists of the sequence set forth in SEQ ID NO:10. ### Claim 32 A composition comprising at least one PCR primer wherein the at least one PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO:13 and further comprises a nucleic acid sequence for use as a barcode and a nucleic acid sequence for use as a sequencing adaptor. ### Claim 33 (depends on 32) The composition of claim 32, wherein the nucleic acid sequence for use as a sequencing adaptor consists of the sequence set forth in SEQ ID NO: 17. ### Claim 34 (canceled) ### Claim 35 (depends on 1) A kit comprising: a) a first composition comprising at least one PCR primer, wherein the at least one PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and further comprises a nucleic acid sequence for use as a sequencing adaptor, and b) a second composition comprising at least one PCR primer wherein the at least one PCR primer comprises a nucleic acid sequence selected from the group comprising SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO:13 and further comprises a nucleic acid sequence for use as a barcode and a nucleic acid sequence for use as a sequencing adaptor, for use in the method of claim 1. ### Claim 36 (depends on 35) The kit of claim 35, further comprising control primers. ### Claim 37 (depends on 36) The kit of claim 36, wherein the control primers have nucleic acid sequences as set forth in SEQ ID NO:27 and SEQ ID NO:28.