A-248 Assessment of Nanopore Sequencing for HIV Genotyping: A Comparative Study with Sanger Sequencing

Abstract

Abstract Background The HIV drug resistance test, conducted through genotyping of the Human Immunodeficiency Virus (HIV), is a critical tool for assessing drug resistance in virus-infected patients and is commonly performed using Sanger Sequencing methodology (first-generation sequencing). Nanopore sequencing (third-generation sequencing) is a new technology that started to be commercialized a few years ago and has recently undergone significant updates with the new Q20+ chemistry. Therefore, this study aims to evaluate the potential use of nanopore sequencing (MinION) for HIV genotyping. Methods The genomic region encoding HIV protease and transcriptase was amplified by nested PCR using primer pairs modified with unique barcodes in the second round of amplification. Three libraries were assembled, including samples from 22 HIV-positive patients, one negative control (negative control patient), and one no-template control (NTC) per library, totaling 66 HIV-positive samples. The libraries were constructed using the SQK-LSK114 kit and sequenced on a MinION platform using the new Q20+ chemistry in flowcells or flongle R10.4.1. The sequences were assembled and analyzed using HIVdb-NGS (Stanford). Validation tests, such as accuracy and precision, were conducted following WHO recommendations for HIV genotyping. The same samples were also sequenced using the Sanger method for comparison. Results 62 HIV sequences were generated by the MinION and analyzed in the described pipeline. The average sequence coverage per sample was 25,866 x. 41 sequences were classified as type B (63.08%), 12 as type C (18.46%), 8 as type F (12.31%), three as CRF02_AG (4.62%), and 1 as a recombinant B+C (1.54%). All identified subtypes were consistent with those found by the Sanger method. The average similarity between the sequences of each sample (MinION vs Sanger) was 98.68% (minimum 94.96%, maximum 99.95%). Basecalling similarity at relevant positions (associated with resistance) was above 98% in 59 out of 62 samples (95.16%), meeting the acceptance criteria recommended by the WHO. Conclusions Our results suggest that nanopore sequencing (utilizing Q20+ chemistry) can replace Sanger sequencing for HIV genotyping, which is used to monitor the effectiveness of anti-HIV drugs. This transition will allow for a reduction in test costs in laboratories with high demand for HIV drug resistance testing. Additionally, the use of modified primers proposed here eliminates the need for commercial barcodes, resulting in reduced processing time and improved throughput.