Kaitlin Reid

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.

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.