Sequencing of the Hepatitis D Virus RNA WHO International Standard

- The WHO Standard for HDV RNA was sequenced by Sanger sequencing and RNA-seq.
- There was 100% agreement between Sanger and RNA-seq consensus sequences.
- The consensus sequence had 89.0% identity to the published WHO sequence.
- The best matching sequence (99.2%) was from the same published study.
- It appears that the identifier and sequence have been incorrectly associated.

BackgroundWell-characterized, stable calibration materials are essential to standardize quantitative viral reporting. The preferred calibration materials are the WHO International Standards and secondary standards derived from them. In 2013, the 1st WHO International Standard for Hepatitis D Virus (HDV) RNA became available. During the course of assay development in our laboratory, differences between the published sequence (GenBank ID: HQ005371) and sequence we generated from the WHO HDV Standard were identified.ObjectivesWe sought to sequence the entire genome of the WHO HDV Standard and compare the results to the published sequence.Study designRNA extracted from the WHO HDV Standard was used to generate five overlapping PCR products, including one covering the entire HDV genome, which were Sanger sequenced using standard dye-terminator chemistry. Total RNA from the WHO HDV Standard was also converted to a cDNA library generating 2.1 million sequencing reads on a NextSeq500 instrument.ResultsSanger sequencing produced 32 overlapping, partial sequences of the HDV genome. RNA-seq resulted in 8100 HDV sequences covering the viral genome an average of 645-fold. Sanger and RNA-seq consensus sequences had 100% agreement and showed 89.0% nucleotide identity with the published WHO HDV Standard sequence. BLAST analysis revealed HQ005369 as the closest match with 99.2% nucleotide identity.ConclusionsHQ005369 was deposited in GenBank along with HQ005371 and seven others from a study of nine Turkish patients. A sample mix-up or clerical error may have resulted in the incorrect association of identifier and sequence. The correct nucleic acid sequence for standards is critical for test accuracy, optimization, calibration, and troubleshooting.