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

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. (c) 2009 Elsevier B.V. All rights reserved.