title: The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool.
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.
authors.affiliation: Department of Medicine, University of California, San Diego, 9500 Gilman Drive, Mail Code 0679, La Jolla, CA 92039, USA. dmbutler@ucsd.edu
authors.forename: David M
authors.initials: DM
authors.lastname: Butler
authors.forename: Mary E
authors.initials: ME
authors.lastname: Pacold
authors.forename: Parris S
authors.initials: PS
authors.lastname: Jordan
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Richman
authors.forename: Davey M
authors.initials: DM
authors.lastname: Smith
mesh_terms: D019295:Computational Biology / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D019562:Viral Load
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral
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