Faculty Mentor: Melodie Weller (Dentistry, University of Utah)

Background

Hepatitis Delta Virus (HDV) is a satellite virus that requires the envelope glycoproteins of a competent helper virus, such as Hepatitis B Virus (HBV), to exit and enter a cell [1]. HDV is characterized in part by its high proportion of self-complementarity and secondary structure forming a densely packed ribonucleoprotein complex, which presents challenges for traditional sequencing methods. We hypothesize that enriching for covalently closed circular RNA (cccRNA) by depleting background RNA using ribonucleases (RNAses), combined with further fragmentation for a smaller average library size, will improve sequencing quality and coverage of the HDV genome.

Methods

293T HEK cells were cultured and transfected with the pSLV D2m plasmid [2] containing two copies of the HDV genome. Total RNA was isolated using RNeasy isolation kits. Linear RNA was degraded using RNase R and ribosomal RNA was depleted using RNase H, enriching for cccRNA. Following RNase treatment, libraries were constructed with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®.

Results

Depletion of linear RNA significantly increased the counts per million (CPM) of HDV reads obtained through Illumina sequencing. This strategy effectively enriched the sample for HDV sequences, with a significant increase in CPM (p<0.05, n=3). As predicted, sequencing coverage showed a notable decrease in quality in the region of HDV’s ribozyme.

Conclusion

Depletion of linear RNA significantly enhanced HDV sequencing quality and coverage. The sequenced region around the ribozyme showed reduced sequencing quality suggesting that the extensive secondary structure in the region may have impeded standard Illumina sequencing. These results support future experiments with different library insert sizes and fragmentation methods to address issues related to HDV’s complex secondary structures. Further optimization is needed for practical application, especially given the low concentrations of HDV RNA in natural infections.

Footnotes

1. Lai, Michael M. C. “The Molecular Biology of Hepatitis Delta Virus.” Annual Review of Biochemistry 64, no. 1 (1995): 259–86. https://doi.org/10.1146/annurev.bi.64.070195.001355.
2. Kuo, M. Y., M. Chao, and J. Taylor. “Initiation of Replication of the Human Hepatitis Delta Virus Genome from Cloned DNA: Role of Delta Antigen.” Journal of Virology 63, no. 5 (May 1989): 1945–50. https://doi.org/10.1128/JVI.63.5.1945-1950.1989.