The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool.

TitleThe efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool.
Publication TypeJournal Article
Year of Publication2009
AuthorsButler DM, Pacold ME, Jordan PS, Richman DD, Smith DM
JournalJ Virol Methods
Volume162
Issue1-2
Pagination280-3
Date Published2009 Dec
ISSN1879-0984
KeywordsComputational Biology, DNA, Complementary, Genome, Viral, HIV Infections, HIV-1, Humans, Poisson Distribution, Polymerase Chain Reaction, RNA, Viral, Sequence Analysis, DNA, Viral Load
Abstract

Typically, population-based sequencing of HIV does not detect minority variants present at levels below 20-30%. Single genome amplification (SGA) and sequencing improves detection, but it requires many PCRs to find the optimal terminal dilution to use. A novel method for guiding the selection of a terminal dilution was developed and compared to standard methods. A quantitative real-time PCR (qRT-PCR) protocol was developed. HIV RNA was extracted, reverse transcribed, and quantitated. A bioinformatics web-based application was created for calculating the optimal concentration of cDNA to use based on results of a trial PCR using the dilution suggested by the qRT-PCR results. This method was compared to the standard. Using the standard protocol, the mean number of PCRs giving an average of 30 (26-34, SD=3) SGA per sample was 245 (218-266, SD=20) after an average of 8 trial dilutions. Using this method, 135 PCRs (135-135, SD=0) produced 30 (27-30, SD=1) SGA using exactly two dilutions. This new method reduced turnaround time from 8 to 2 days. Standard methods of SGA sequencing can be costly and both time- and labor-intensive. By choosing a terminal dilution concentration with the proposed method, the number of PCRs required is decreased and efficiency improved.

DOI10.1016/j.jviromet.2009.08.002
PubMed URLhttp://www.ncbi.nlm.nih.gov/pubmed/19698751?dopt=Abstract
PMCPMC2761514
Alternate JournalJ. Virol. Methods
PubMed ID19698751