Background
Choosing a next-generation sequencing (NGS) workflow to enable sensitive and rapid detection of infectious agents remains critical. This collaborative study compared multiple NGS methods to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load.
Methods
23 HBV DNA-positive plasma samples of genotypes A-E (0.2 to 6207 IU/ml) and one control sample were assayed blindly via 9 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. Construction of consensus sequences was performed at the coordinating centre.
Results
Full HBV genomes were constructed at viral loads >1000 IU/ml for probe-capture methods, >200 IU/ml for PCR-Illumina methods, >10 IU/ml for PCR-Nanopore methods, and in no samples for metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads for PCR-based methods. Probe-capture and metagenomic methods detected additional viruses not routinely screened in blood donations; positive results were confirmed by PCR. Costs were lowest for PCR-Nanopore, and turnaround time was highest for probe-capture methods.
Conclusion
Different methods have different advantages, and the optimal method depends on the context. NGS has the potential to delineate whole-genome sequences at low viral loads if supported by a PCR pre-amplification step. Probe-capture methods also reliably detect HBV at low viral loads but limit genome characterisation while accommodating the incidental detection of other virus species. Stringent steps are needed to prevent cross-contamination or bioinformatic noise to maximise diagnostic accuracy.
Importance
There is a great need in clinical microbiology and public health to sequence whole genomes of different viruses. Because so many different methods can be used for sequencing, it can be difficult to choose a suitable method. We compared commonly used methods on blood donor samples with low levels of hepatitis B virus DNA. We found that each method had its own benefits; however, our study highlights the need for increased vigilance regarding the risk of contamination. Our study provides necessary data for clinical and research laboratory teams to make informed decisions about the most suitable method. This would advance our understanding of hepatitis B and other viruses, ultimately benefiting both current and future patients.