Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing
AbstractGenetic analyses play a central role in infectious disease research. Massively parallelized “mechanical cloning” and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure—from nucleic acid extraction to sequencing—should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping. View Full-Text
- Supplementary File 1:
Supplementary (ZIP, 2267 KB)
Share & Cite This Article
Vrancken, B.; Trovão, N.S.; Baele, G.; van Wijngaerden, E.; Vandamme, A.-M.; van Laethem, K.; Lemey, P. Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing. Viruses 2016, 8, 12.
Vrancken B, Trovão NS, Baele G, van Wijngaerden E, Vandamme A-M, van Laethem K, Lemey P. Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing. Viruses. 2016; 8(1):12.Chicago/Turabian Style
Vrancken, Bram; Trovão, Nídia S.; Baele, Guy; van Wijngaerden, Eric; Vandamme, Anne-Mieke; van Laethem, Kristel; Lemey, Philippe. 2016. "Quantifying Next Generation Sequencing Sample Pre-Processing Bias in HIV-1 Complete Genome Sequencing." Viruses 8, no. 1: 12.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.