Your browser doesn't support javascript.
loading
Sequencing of the Hepatitis D Virus RNA WHO International Standard.
Pyne, M T; Mallory, M A; Xie, H B; Mei, Y; Schlaberg, R; Hillyard, D R.
Afiliação
  • Pyne MT; ARUP Laboratories, Salt Lake City, UT, USA. Electronic address: michael.pyne@aruplab.com.
  • Mallory MA; ARUP Laboratories, Salt Lake City, UT, USA.
  • Xie HB; IDbyDNA, San Francisco, CA, USA.
  • Mei Y; IDbyDNA, San Francisco, CA, USA.
  • Schlaberg R; ARUP Laboratories, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA.
  • Hillyard DR; ARUP Laboratories, Salt Lake City, UT, USA; Department of Pathology, University of Utah, Salt Lake City, UT, USA.
J Clin Virol ; 90: 52-56, 2017 05.
Article em En | MEDLINE | ID: mdl-28359844
ABSTRACT

BACKGROUND:

Well-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.

OBJECTIVES:

We sought to sequence the entire genome of the WHO HDV Standard and compare the results to the published sequence. STUDY

DESIGN:

RNA 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.

RESULTS:

Sanger 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.

CONCLUSIONS:

HQ005369 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.
Assuntos
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Padrões de Referência / RNA Viral / Vírus Delta da Hepatite / Genoma Viral / Análise de Sequência de DNA Limite: Humans Idioma: En Revista: J Clin Virol Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Padrões de Referência / RNA Viral / Vírus Delta da Hepatite / Genoma Viral / Análise de Sequência de DNA Limite: Humans Idioma: En Revista: J Clin Virol Ano de publicação: 2017 Tipo de documento: Article