The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool.
|Title||The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool.|
|Publication Type||Journal Article|
|Year of Publication||2009|
|Authors||Butler DM, Pacold ME, Jordan PS, Richman DD, Smith DM|
|Journal||J Virol Methods|
|Date Published||2009 Dec|
|Keywords||Computational Biology, DNA, Complementary, Genome, Viral, HIV Infections, HIV-1, Humans, Poisson Distribution, Polymerase Chain Reaction, RNA, Viral, Sequence Analysis, DNA, Viral Load|
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.
|Alternate Journal||J. Virol. Methods|