Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes.
Methods Mol Biol
; 1746: 55-61, 2018.
Article
en En
| MEDLINE
| ID: mdl-29492886
ABSTRACT
We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project, which analyzes complete viral genomes from more than 3000 HIV-1-infected Europeans with high-throughput deep sequencing techniques. The goal of the BEEHIVE project is to determine the contribution of viral genetics to virulence. Recently we performed a pilot experiment with 125 patient plasma samples to identify the method that is most suitable for isolation of HIV-1 viral RNA for subsequent long-amplicon deep sequencing. We reported that manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) provides superior results over robotically extracted RNA. The latter approach used the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics), the QIAcube robotic system (Qiagen), or the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular). Here we present a detailed protocol for the labor-intensive manual extraction method that yielded the best results.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
ADN Viral
/
Infecciones por VIH
/
VIH-1
/
Genoma Viral
/
Secuenciación de Nucleótidos de Alto Rendimiento
Tipo de estudio:
Diagnostic_studies
/
Guideline
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2018
Tipo del documento:
Article
País de afiliación:
Países Bajos