Next-generation sequencing methods for sensitive hepatitis B viral genome analysis: A European study

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Michael X Fu Maria F Perdomo Sheila F Lumley Johan Ringlander Kai Kean Kaitlin Reid Richard Mayne Oscar Enrique Torres Montaguth Leysa Forrest Sarah Buddle Johannes C Botha Joakim B Stenbäck Zachery Dickson Chris Kent Haiting Chai Matthew Byott Leo Hannolainen Shannah Secret George Airey Klaus Hedman Monique I Andersson M Azim Ansari Eleni Nastouli Judith Breuer Philippa Matthews Tanya Golubchik William L Irving Peter Simmonds Heli Harvala
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Abstract

This multicentre study investigated the utility of next-generation sequencing (NGS) to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load (from 0.2 to 6207 IU/mL). 23 HBV DNA-positive plasma samples of genotypes A-E and one HBV-negative control sample were assayed blindly via 9 established NGS methods from 6 European laboratories. Methods included untargeted metagenomics, pre-enrichment by probe-capture followed by Illumina sequencing, and HBV-specific PCR pre-amplification followed by sequencing with Nanopore or Illumina. Full HBV genomes were obtained only from samples with viral loads > 1000 IU/mL using probe-capture methods, > 200 IU/mL using PCR-Illumina methods, > 10 IU/mL using PCR-Nanopore methods, and in no samples using metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads in PCR-based methods. Probe-capture and metagenomic methods detected additional viruses not routinely screened in blood donations, including polyomaviruses and herpesviruses; positive results were confirmed by PCR. In conclusion, NGS may delineate whole-genome sequences at low viral loads if supported by a PCR pre-amplification step. Probe-capture methods also reliably detect HBV without pre-amplification but show limited genome coverage for samples with low viral loads; they may additionally detect a wide range of blood-borne viruses.

Journal details

Journal Journal of Medical Virology
Volume 98
Issue number 9
Pages e71130
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Publisher website (DOI) 10.1002/jmv.71130
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Europe PubMed Central 42683603
Pubmed 42683603

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The publication was previously a preprint. View preprint

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