Developing an Accurate Assay for Detection of Serum HBV RNA and SP1 Variant in CHB Based on Long‐Read Sequencing

The profiles of spliced pgRNA variants in NAs-treated HBV-infected patients were still unclear, and the sensitivity and accuracy of quantification of HBV RNA or spliced pgRNA variants 1 (SP1) needs improvement. In this study, we identified the pattern of spliced pgRNA variants in HBV-infected patients including treatment-naïve and NAs-treatment with our established long-read sequencing strategy. 16 reported spliced pgRNA and 41 novel spliced variants were identified by long-read sequencing, and SP1 variant was the most abundant among all HBV spliced RNA, ranging from 35% to 62%. The proportion of spliced pgRNA/total HBV RNA and SP1/HBV RNA decreased after short-term NAs treatment. Furthermore, we established an accurate assay for detection of serum HBV RNA and SP1 based on long-read sequencing results, and evaluated the detection capability in HBV-infected patients. A good consistency was observed in quantification of serum HBV RNA levels both in treatment-naïve and NAs treatment patients measured by primers sets 2312/2413 and 1528/1600 located outside the SP1 region. Finally, the proportion of SP1/HBV RNA was associated with the degree of liver inflammation. Therefore, we determined the characteristics of spliced pgRNA and dynamic change in NAs-treated patients by long-read sequencing, and provide more information to support developing more accurate HBV RNA or SP1 variant detection assay.