Nanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typing

Abstract

Background: Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50% of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistance-associated substitutions (RAS) is not practical in many locations.Objectives: A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing. Study design:The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2x10 8 to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer. Results:The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2kb amplicon using a MinION sequencer gave results consistent with Sanger and allowed the identification of minor populations of variants. Conclusions:We present two methods for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses.

Hepatitis B in the Northwestern region of Sao Paulo State: genotypes and resistance mutations

“…Astbury et al 5 conducted the only HBV study in the region of Sao Jose do Rio Preto, and identified the subgenotypes A1, A2, B1, D1, D2, D3, F1 and F2. In the present study, we detected the same subgenotypes but we collected a larger number of samples in a much larger area of the region.…”

“…Astbury et al 5 conducted the only study of HBV genotyping and resistance mutations analysis in Sao Jose do Rio Preto. They identified the subgenotypes A1, A2, B1, D1, D2, D3, F1 and F2, in addition to strains resistant to the nucleoside analogues (NA) lamivudine (LAM), entecavir (ETV), and telbivudine (LDT).…”

Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

“…Ultra-long reads are more likely to cover the complete viral genome, highly repetitive regions, and structural variations in human genome, allowing continuous and complete genome assembly ( Jain et al, 2018 ; Senol Cali et al, 2018 ). With their long reading capacity, nanopore sequencing technologies have shown great advantages in the rapid surveillance of Ebola ( Hoenen, 2016 ; Quick et al, 2016 ) and Zika viruses ( Quick et al, 2017 ), genotyping and genetic diversity analysis of hepatitis B virus (HBV) ( Sauvage et al, 2018 ; McNaughton et al, 2019 ; Astbury et al, 2020 ), and monitoring emerging infectious disease outbreaks ( Zhu et al, 2020 ). Whole genome sequencing (WGS) of SARS-CoV-2 with nanopore sequencing technologies provided a unique tool to analyze viral transmission, evolution, and genomic variation ( Bull et al, 2020 ; Lin et al, 2021 ).…”

Concurrent Nanopore Next-Generation Sequencing of Hepatitis B and Delta Virus Genomes Directly From Patient Plasma

“…The advent of next-generation sequencing (NGS) techniques has considerably improved the detection and characterization of microbial and viral genetic sequences. Although initially used to study environmental, animal, and human samples, NGS has been more recently introduced in clinical microbiology and virology laboratories [ 1 – 5 ]. Its technical requirements, processing time, and cost are now compatible with diagnostic use.…”

“…We report for the first time, to the best of our knowledge, the concurrent detection and determination of HBV and HDV sequences, including full HBV genome recovery, on the same day and directly from nucleic acids extracted from plasma. Illumina or ONT NGS has been previously used to obtain HBV or HDV sequences, but generally only after target enrichment, which is more time-consuming [ 2 , 9 ]. NGS directly from DNA or RNA extract without PCR amplification, as was done in the present case, is simpler and faster and allows simultaneous detection of several non-targeted infectious agents.…”