Presentation description

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. HDV is characterized in part by its high proportion of self-complementarity and secondary structure forming a densely packed RNA genome, which presents challenges for traditional sequencing methods. We hypothesize that enriching circular RNA and depleting linear RNA, combined with fragmenting for a smaller average library size, will improve sequencing quality and coverage when targeting the HDV genome and its transcripts.

Methods

293T HEK cells were cultured and transfected with a plasmid containing the HDV genome (pSVLD2m). 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 circular RNA. Following RNase treatment, libraries were constructed with the NEBNext Ultra II Directional RNA Library Prep Kit.

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 (paired t test; p<0.05, n=3). Sequencing coverage showed a notable dip around the ribozyme region on the HDV genome.

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.