Dual sequence-capture method for quantifying trans renal HPV DNA in urine

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.

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.