Quantitative characterization of hepatitis delta virus genome edition by next-generation sequencing.

Aim: To determine the capacity of next-generation sequencing (NGS) for quantifying edited and unedited HDV populations, and to confirm if edition is a general phenomenon taking place along the entire HDV region analyzed, as we previously reported (Homs M et al. PLoS One 2016, 11, e0158557).

Methods: Four serum samples from 4 patients with chronic HDV/HBV infection were included in the study. The region selected for analysis covered 360 nucleotides (nt), positions 910-1270 of the HDV genome, which included the HDAg ORF editing site (nt 1014 within codon 196). Quantification of edited and unedited genomes was performed by molecular cloning and Sanger sequencing and by NGS. To evaluate the reliability of the NGS values obtained, we combined a clone with an edited codon and one with an unedited codon in known percentages in a series of artificial mixtures, which were then analyzed by NGS. In addition, we determined the nt changes occurring over the complete amplified region after excluding the editing codon (196) to evaluate edition along it.

Results: In total, 11,208 quality-filtered sequences were obtained in the 4 samples. The 95% confidence intervals for the proportions of unedited populations by molecular cloning and NGS were overlapping, and those of cloning were wider, indicating that they are comparable and that NGS is more precise than cloning. Unedited genomes predominated over edited ones in all 4 samples analyzed by NGS and in 3 of the 4 samples analyzed by molecular cloning. In total, 83,276 quality-filtered sequences were obtained from the artificial mixtures. Percentages of the two viral populations detected by NGS in these mixtures were comparable to the expected percentages. Evaluation of edition along the HDV coding region showed that transitions were more frequent than transversions, accounting for 63.09% and 36.91%, respectively. Interestingly, among the 4 possible transition-type changes, G:A and A:G accounted for 73.86% of the total.

Conclusion: Next-generation sequencing proved useful to quantify edited and unedited HDV genomes, and provided relevant information on the HDV quasispecies.