High-dimensional droplet digital PCR of multiple hepatitis B splice variants in serum, liver, and tissue culture

Hepatitis B virus (HBV) affects approximately 2 billion individuals; 254 million have chronic infection. The roles of spliced HBV (spHBV) RNAs in human infection are poorly understood partly due to limitations in quantitative methods. We designed and multiplexed long amplicon (0.3–2kbp) ddPCR assays that simultaneously quantify the most frequently observed spHBV isoforms (sp1, sp2, sp3, sp8, sp9) alongside unspliced pregenomic RNA (pgRNA). Our innovations include the use of ddPCR, long amplicons, and high-dimensional multiplexing. We tested primers and probes using qPCR on synthetic DNA targets to identify a high-fidelity combination recognizing HBV genotype A viruses. Using common forward and reverse primers we accounted for PCR efficiency differences between amplicons of differing size. We altered denaturation and annealing temperatures to enrich spHBV, the minority population of total HBV RNA. To achieve higher-order multiplexing, we increased the number of cycles to 80, added a post-reverse transcription cleanup step, and tested > 360 oligo concentration combinations. We performed spatial transcriptomic sequencing on liver tissue to confirm splice junctions measured by ddPCR. We successfully quantified spHBV and pgRNA in serum, liver, and HBV-infected HepG2^NTCP cells. Our assay enables direct, sensitive quantification of spHBV and full-length pgRNA from serum, liver, and tissue culture samples, which will facilitate studies on the impact of spHBV on HBV infection outcomes in people. This technique has potential applications in HBV research and therapy development, with broader implications in virology and oncology.