Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
St John, EP; Simen, BB; Turenchalk, GS; Braverman, MS; Abbate, I; Aerssens, J; Bouchez, O; Gabriel, C; Izopet, J; Meixenberger, K; Di Giallonardo, F; Schlapbach, R; Paredes, R; Sakwa, J; Schmitz-Agheguian, GG; Thielen, A; Victor, M; Metzner, KJ; Däumer, MP; 454 HIV-1 Alpha Study Group.
A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.
PLoS One. 2016; 11(1):e0146687-e0146687
Doi: 10.1371/journal.pone.0146687
[OPEN ACCESS]
Web of Science
PubMed
FullText
FullText_MUG
- Co-Autor*innen der Med Uni Graz
- GABRIEL Christian
- Altmetrics:
- Dimensions Citations:
- Plum Analytics:
- Scite (citation analytics):
- Abstract:
- Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940-447,400 copies/mL, two dilution series (52,129-1,340 and 25,130-734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10-99, RT codons 1-251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592-3,488) and 2,410 for V3 (IQR 786-3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2-5.2 ± 0.04-0.65). In the two dilutions series, no variants >20% were missed, variants 2-10% were detected at most sites (even at low VT), and variants 1-2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.
- Find related publications in this database (using NLM MeSH Indexing)
- Amino Acids - genetics
- Cooperative Behavior -
- Drug Resistance, Viral - genetics
- Follow-Up Studies -
- HIV-1 - genetics
- High-Throughput Nucleotide Sequencing - methods
- Humans -
- Mutation - genetics
- Plasmids - genetics
- Reproducibility of Results -
- Tropism - genetics