HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Human papillomavirus (HPV) genotyping is crucial for cervical cancer screening, often combined with high-risk (HR) HPV detection.
• Existing methods like L1 sequencing and APTIMA HPV genotyping have limitations in comprehensive HPV profiling.

Purpose of the Study:

• To develop and evaluate a novel HPV genotyping method utilizing PacBio single molecule real-time (SMRT) sequencing of the E6/E7 region.
• To assess the performance of this new method against established techniques (MY09-11 L1 sequencing and APTIMA HPV genotyping).

Main Methods:

• PacBio SMRT sequencing was employed to analyze approximately 800 bp sequences in the E6/E7 region of HPV.
• The performance of PacBio E6/E7 SMRT genotyping was compared with MY09-11 L1 sequencing and the APTIMA HPV genotyping assay.

Main Results:

• PacBio E6/E7 SMRT sequencing demonstrated 100% concordance with MY09-11 L1 sequencing and 90.8% concordance with APTIMA HPV genotyping.
• The sensitivity of PacBio E6/E7 SMRT was comparable to L1 sequencing and lower than HR-HPV tests, as expected.
• PacBio SMRT sequencing offers unbiased genotyping, capable of detecting multiple HPV infections and intra-genotype variations (haplotypes).

Conclusions:

• PacBio E6/E7 SMRT sequencing is a robust and accurate method for both high-risk (HR) and low-risk (LR) HPV genotyping in clinical samples.
• This technique is particularly useful as a follow-up genotyping assay after a positive HPV test in cervical cancer screening protocols.
• The ability to detect multiple infections and haplotypes makes PacBio SMRT sequencing a valuable advancement in HPV diagnostics.