US 9,809,864 B2
Dual sequence-capture method for quantifying trans renal HPV DNA in urine
Rafael Guerrero Preston, Baltimore, MD (US); Anne Jedlicka, Fallston, MD (US); and David Sidransky, Baltimore, MD (US)
Assigned to THE JOHNS HOPKINS UNIVERSITY, Baltimore, MD (US)
Appl. No. 14/772,004
Filed by THE JOHNS HOPKINS UNIVERSITY, Baltimore, MD (US)
PCT Filed Mar. 3, 2014, PCT No. PCT/US2014/019934
§ 371(c)(1), (2) Date Sep. 1, 2015,
PCT Pub. No. WO2014/134607, PCT Pub. Date Sep. 4, 2014.
Claims priority of provisional application 61/771,462, filed on Mar. 1, 2013.
Prior Publication US 2016/0010163 A1, Jan. 14, 2016
Int. Cl. C12Q 1/70 (2006.01); C12Q 1/68 (2006.01)
CPC C12Q 1/708 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6886 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/154 (2013.01)] 15 Claims
 
1. A sequence-based method for detecting HPV Trans-Renal DNA (TrDNA) in a subject, the method comprising:
(a) isolating one or more low molecular weight, fragmented cell-free nucleic acids from a urine sample from a subject thereby creating a library of the low molecular weight, fragmented cell-free nucleic acids;
(b) enriching the one or more low molecular weight fragmented cell-free nucleic acids isolated from the urine sample for HPV TrDNA using a high-risk HPV-specific solution-based capture method to enrich the HPV genome to produce one or more enriched HPV TrDNA, wherein enriching the one or more low molecular weight fragmented cell-free nucleic acids comprises:
(i) amplifying the HPV TrDNA in the library using ligation-mediated PCR (LM-PCR) to form a pre-capture PCR library;
(ii) hybridizing the pre-capture PCR library to a pool of HPV-specific capture probes specific for the HPV E1 region of the following HPV subtypes selected from the group consisting of: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, to form a post-capture PCR library;
(iii) amplifying the post-capture PCR library to produce one or more enriched HPV TrDNA; and
(iv) optionally repeating steps (iii) and (iv);
(d) adding at least one index to the one or more enriched HPV TrDNA;
(e) performing multiplexed sequencing of the one or more enriched HPV TrDNA having at least one index added thereto to produce a multiplexed nucleotide sequence;
(f) performing a sequence alignment between the multiplexed nucleotide sequence and the nucleotide sequence of one or more known HPV genotypes; and
(g) determining the percentage sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the one or more known HPV genotypes;
wherein at least a 60% sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the one or more known HPV genotypes means that HPV TrDNA has been detected in the subject.
 
9. A sequence-based method for predicting or screening for cancer by detecting high-risk HPV Trans-Renal DNA (TrDNA) in a subject, the method comprising:
(a) isolating one or more low molecular weight, fragmented cell-free nucleic acids from a urine sample from a subject thereby creating a library of the low molecular weight, fragmented cell-free nucleic acids;
(b) enriching the one or more low molecular weight fragmented cell-free nucleic acids isolated from the urine sample for HPV TrDNA using a high-risk HPV-specific solution-based capture method to enrich the HPV genome to produce one or more enriched HPV TrDNA, wherein enriching the one or more low molecular weight fragmented cell-free nucleic acids comprises:
(i) amplifying the HPV TrDNA in the library using ligation-mediated PCR (LM-PCR) to form a pre-capture PCR library;
(ii) hybridizing the pre-capture PCR library to a pool of HPV-specific capture probes specific for the HPV E1 region of the following HPV subtypes selected from the group consisting of: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, to form a post-capture PCR library;
(iii) amplifying the post-capture PCR library to produce one or more enriched HPV TrDNA; and
(iv) optionally repeating steps (iii) and (iv);
(d) adding at least one index to the one or more enriched HPV TrDNA;
(e) performing multiplexed sequencing of the one or more enriched HPV TrDNA having at least one index added thereto to produce a multiplexed nucleotide sequence;
(f) performing a sequence alignment between the multiplexed nucleotide sequence and the nucleotide sequence of one or more known HPV genotypes; and
(g) determining the percentage sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the one or more known HPV genotypes;
wherein at least a 60% sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the one or more known high-risk HPV genotypes is indicative that the subject has or is at risk for developing a cancer.
 
15. A method for detecting methylated human Trans-Renal DNA (TrDNA) in a subject, the method comprising:
(a) isolating one or more low molecular weight, fragmented cell-free nucleic acids from a urine sample from a subject thereby creating a library of the low molecular weight, fragmented cell-free nucleic acids;
(b) enriching the one or more low molecular weight fragmented cell-free nucleic acids isolated from the urine sample for HPV TrDNA using a high-risk HPV-specific solution-based capture method to enrich the HPV genome to produce one or more enriched HPV TrDNA, wherein enriching the one or more low molecular weight fragmented cell-free nucleic acids comprises:
(i) amplifying the HPV TrDNA in the library using ligation-mediated PCR (LM-PCR) to form a pre-capture PCR library;
(ii) hybridizing the pre-capture PCR library to a pool of HPV-specific capture probes specific for the HPV E1 region of the following HPV subtypes selected from the group consisting of: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, to form a post-capture PCR library;
(iii) amplifying the post-capture PCR library to produce one or more enriched HPV TrDNA; and
(iv) optionally repeating steps (iii) and (iv);
(d) adding at least one index to the one or more enriched human TrDNA;
(e) performing multiplexed sequencing of the one or more enriched human TrDNA having at least one index added thereto to produce a multiplexed nucleotide sequence;
(f) performing a sequence alignment between the multiplexed nucleotide sequence and the nucleotide sequence of the human genome; and
(g) determining the percentage sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the human genome;
wherein prior to step (d) the post-capture library is treated with a bisulfite compound and is amplified using PCR to form one or more amplified methylated human TrDNA; and further wherein at least a 60% sequence identity between the multiplexed nucleotide sequence and the nucleotide sequence of the human genome means that methylated human TrDNA has been detected in the subject.