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

ABSTRACT 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.