Whole genome sequencing of hepatitis B virus using tiled amplicon (HEPTILE) and probe based enrichment on Illumina and Nanopore platforms
Abstract
Abstract Hepatitis B virus (HBV) whole genome sequencing (WGS) is currently limited as the DNA viral loads (VL) of many clinical samples are below the threshold required to generate full genomes using current sequencing methods. We developed two pan-genotypic viral enrichment methods, using probe-based capture and tiled amplicon PCR (HEP-TILE) for HBV WGS. We demonstrate using mock samples that both enrichment methods are pan-genotypic (genotypes A-J). Using clinical samples, we demonstrate that HEP-TILE amplification successfully amplifies full genomes at the lowest HBV VL tested (30 IU/ml), and the PCR products can be sequenced using both Nanopore and Illumina platforms. Probe-based capture with Illumina sequencing required VL > 300,000 IU/ml to generate full length HBV genomes. The capture-Illumina and HEP-TILE-Nanopore pipelines had consensus sequencing accuracy of 100% in mock samples with known DNA sequences. Together, these protocols will facilitate the generation of HBV sequence data, enabling a more accurate and representative picture of HBV molecular epidemiology, cast light on persistence and pathogenesis, and enhance understanding of the outcomes of infection and its treatment.
Article Details
Authors (30)
Sheila F. Lumley
Chris Kent
Daisy Jennings
Haiting Chai
George Airey
Elizabeth Waddilove
Marion Delphin
Amy Trebes
Anna L. McNaughton
Khadija Said Mohammed
Sam A. J. Wilkinson
Yanxia Wu
George MacIntyre-Cockett
Beatrice Kimono
Kwizera Moses Mbonye
Kevin Ojambo
Tongai G. Maponga
Cedric C. S. Tan
Catherine de Lara
Jacqueline Martin
James Campbell
Marije Van Schalkwyk
Dominique Goedhals
Robert Newton
Eleanor Barnes
Nicholas J. Loman
Paolo Piazza
Joshua Quick
M. Azim Ansari
Philippa C. Matthews