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Objectives

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).

Methods

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.

Results

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 all 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.

Conclusions

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 achieve limited genome characterisation at low viral loads; they may additionally detect a wide range of blood-borne viruses.