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.
Butler
David M
DM
Department of Medicine, University of California, San Diego, 9500 Gilman Drive, Mail Code 0679, La Jolla, CA 92039, USA. dmbutler@ucsd.edu
Pacold
Mary E
ME
Jordan
Parris S
PS
Richman
Douglas D
DD
Smith
Davey M
DM
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
D018076:DNA, Complementary; D012367:RNA, Viral
J Infect Dis. 2004 Nov 15;190(10):1880-8
15499546
AIDS Res Hum Retroviruses. 1998 Jul 1;14(10):869-76
9671215
J Virol. 2008 Apr;82(8):3952-70
18256145
Nucleic Acids Res. 1990 Apr 11;18(7):1687-91
2186361
J Virol Methods. 1998 Dec;76(1-2):139-48
9923748
J Virol. 1990 Feb;64(2):864-72
2296085
J Clin Microbiol. 2005 Jan;43(1):406-13
15635002
AIDS Res Hum Retroviruses. 1996 Mar 1;12(4):303-6
8906990
Genome Res. 1996 Oct;6(10):986-94
8908518
Genetics. 2004 Aug;167(4):1573-83
15342499
PLoS Med. 2008 Jul 29;5(7):e158
18666824
J Virol. 1990 Dec;64(12):5840-50
2243378