PCR Amplification Strategies Towards Full-length HIV-1 Genome Sequencing

Author(s): Chao Chun Liu, Hezhao Ji*

Journal Name: Current HIV Research

Volume 16 , Issue 2 , 2018

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Graphical Abstract:


The advent of next-generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of the published protocols were reviewed.

Keywords: HIV-1, Full genome, PCR, Methods, Sequencing, Next generation sequencing.

Chin BS. Molecular Epidemiology of Human Immunodeficiency Virus. Infect Chemother 2017; 49(1): 1-9.
Grossmann S, Nowak P, Neogi U. Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical management. J Int AIDS Soc 2015; 18: 20035.
Yebra G, Hodcroft EB, Ragonnet-Cronin ML, et al. Using nearly full-genome HIV sequence data improves phylogeny reconstruction in a simulated epidemic. Sci Rep 2016; 6: 39489.
Stecher M, Chaillon A, Eberle J, et al. Molecular Epidemiology of the HIV Epidemic in Three German Metropolitan Regions - Cologne/Bonn, Munich and Hannover, 1999-2016. Sci Rep 2018; 8(1): 6799.
Dolling D, Sabin C, Delpech V, et al. Time trends in drug resistant HIV-1 infections in the United Kingdom up to 2009: multicentre observational study. BMJ 2012; 345: e5253.
Frentz D, Van de Vijver DA, Abecasis AB, et al. Increase in transmitted resistance to non-nucleoside reverse transcriptase inhibitors among newly diagnosed HIV-1 infections in Europe. BMC Infect Dis 2014; 14: 407.
Lee GQ, Bangsberg DR, Mo T, et al. Prevalence and clinical impacts of HIV-1 intersubtype recombinants in Uganda revealed by near-full-genome population and deep sequencing approaches. AIDS 2017; 31(17): 2345-54.
Wheeler WH, Ziebell RA, Zabina H, et al. Prevalence of transmitted drug resistance associated mutations and HIV-1 subtypes in new HIV-1 diagnoses, U.S.-2006. AIDS 2010; 24(8): 1203-12.
Novitsky V, Zahralban-Steele M, McLane MF, et al. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering. J Clin Microbiol 2015; 53(8): 2581-92.
Cote HC, Brumme ZL, Harrigan PR. Human immunodeficiency virus type 1 protease cleavage site mutations associated with protease inhibitor cross-resistance selected by indinavir, ritonavir, and/or saquinavir. J Virol 2001; 75(2): 589-94.
Malet I, Roquebert B, Dalban C, et al. Association of Gag cleavage sites to protease mutations and to virological response in HIV-1 treated patients. J Infect 2007; 54(4): 367-74.
Parikh UM, McCormick K. van ZG Future technologies for monitoring HIV drug resistance and cure. Curr Opin HIV AIDS 2017; 12(2): 182-9.
Alampalli SV, Thomson MM, Sampathkumar R, et al. Deep sequencing of near full-length HIV-1 genomes from plasma identifies circulating subtype C and infrequent occurrence of AC recombinant form in Southern India. PLoS One 2017; 12(12): e0188603.
Yebra G, Frampton D, Gallo CT, et al. A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project. PLoS One 2018; 13(2): e0192081.
HIV Sequence Compendium 2017. Los Alamos, New Mexico: Los Alamos National Laboratory, Theoretical Biology and Biophysics 2017.
Liang B, Luo M, Scott-Herridge J, et al. A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1. PLoS One 2011; 6(10): e26745.
Moscona R, Ram D, Wax M, et al. Comparison between next-generation and Sanger-based sequencing for the detection of transmitted drug-resistance mutations among recently infected HIV-1 patients in Israel, 2000-2014. J Int AIDS Soc 2017; 20(1): 21846.
Cornelissen M, Gall A, Vink M, et al. From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing. Virus Res 2017; 239: 10-6.
Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 2016; 17(6): 333-51.
Gall A, Ferns B, Morris C, et al. Universal amplification, next-generation sequencing, and assembly of HIV-1 genomes. J Clin Microbiol 2012; 50(12): 3838-44.
Henn MR, Boutwell CL, Charlebois P, et al. Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection. PLoS Pathog 2012; 8(3): e1002529.
Sigaloff KC, Ramatsebe T, Viana R, et al. Accumulation of HIV drug resistance mutations in patients failing first-line antiretroviral treatment in South Africa. AIDS Res Hum Retroviruses 2012; 28(2): 171-5.
Zanini F, Brodin J, Albert J, et al. Error rates, PCR recombination, and sampling depth in HIV-1 whole genome deep sequencing. Virus Res 2017; 239: 106-14.
Boltz VF, Rausch J, Shao W, et al. Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA. Retrovirology 2016; 13(1): 87.
Palmer S, Kearney M, Maldarelli F, et al. Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis. J Clin Microbiol 2005; 43(1): 406-13.
Salazar-Gonzalez JF, Bailes E, Pham KT, et al. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J Virol 2008; 82(8): 3952-70.
van ZG, Bale MJ, Kearney MF. HIV evolution and diversity in ART-treated patients. Retrovirology 2018; 15(1): 14.
Wang XQ, Palmer S. Single-molecule techniques to quantify and genetically characterise persistent HIV. Retrovirology 2018; 15(1): 3.
Dilernia DA, Chien JT, Monaco DC, et al. Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing. Nucleic Acids Res 2015; 43(20): e129.
Ashton PM, Nair S, Dallman T, et al. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat Biotechnol 2015; 33(3): 296-300.
Nagarajan N, Pop M. Sequence assembly demystified. Nat Rev Genet 2013; 14(3): 157-67.
Lu H, Giordano F, Ning Z. Oxford Nanopore MinION Sequencing and Genome Assembly. Genomics Proteomics Bioinformatics 2016; 14(5): 265-79.
Giallonardo FD, Topfer A, Rey M, et al. Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations. Nucleic Acids Res 2014; 42(14): e115.
Beerenwinkel N, Gunthard HF, Roth V, et al. Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data. Front Microbiol 2012; 3: 329.
Berg MG, Yamaguchi J, Alessandri-Gradt E, et al. A Pan-HIV Strategy for Complete Genome Sequencing. J Clin Microbiol 2016; 54(4): 868-82.
Di GF, Zagordi O, Duport Y, et al. Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination. PLoS One 2013; 8(9): e74249.
Ode H, Matsuda M, Matsuoka K, et al. Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq. Front Microbiol 2015; 6: 1258.
Zanini F, Brodin J, Thebo L, et al. Population genomics of intrapatient HIV-1 evolution. eLife 2015; 4.
Garcia-Arriaza J, Domingo E, Briones C. Characterization of minority subpopulations in the mutant spectrum of HIV-1 quasispecies by successive specific amplifications. Virus Res 2007; 129(2): 123-34.
Paredes R, Clotet B. Clinical management of HIV-1 resistance. Antiviral Res 2010; 85(1): 245-65.
Yin L, Liu L, Sun Y, et al. High-resolution deep sequencing reveals biodiversity, population structure, and persistence of HIV-1 quasispecies within host ecosystems. Retrovirology 2012; 9: 108.
Zagordi O, Klein R, Daumer M, et al. Error correction of next-generation sequencing data and reliable estimation of HIV quasispecies. Nucleic Acids Res 2010; 38(21): 7400-9.
Basu C. PCR primer design. New York: Humana Press 2015.
Brodin J, Krishnamoorthy M, Athreya G, et al. A multiple-alignment based primer design algorithm for genetically highly variable DNA targets. BMC Bioinformatics 2013; 14: 255.
Yoon H, Leitner T. PrimerDesign-M: a multiple-alignment based multiple-primer design tool for walking across variable genomes. Bioinformatics 2015; 31(9): 1472-4.
Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 2002; 16(1): 47-51.
Holleley CE, Geerts PG. Multiplex Manager 1.0: a cross-platform computer program that plans and optimizes multiplex PCR. Biotechniques 2009; 46(7): 511-7.
Giallonardo FD, Topfer A, Rey M, et al. Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations. Nucleic Acids Res 2014; 42(14): e115.
ALPF Medical Research. In: 2017.
Ratmann O, Wymant C, Colijn C, et al. HIV-1 full-genome phylogenetics of generalized epidemics in sub-Saharan Africa: impact of missing nucleotide characters in next-generation sequences. AIDS Res Hum Retroviruses 2017. [Epub ahead of print].
Mild M, Hedskog C, Jernberg J, et al. Performance of ultra-deep pyrosequencing in analysis of HIV-1 pol gene variation. PLoS One 2011; 6(7): e22741.
Jabara CB, Jones CD, Roach J, et al. Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc Natl Acad Sci USA 2011; 108(50): 20166-71.
Shao W, Boltz VF, Spindler JE, et al. Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA. Retrovirology 2013; 10: 18.
van Dijk EL, Jaszczyszyn Y, Thermes C. Library preparation methods for next-generation sequencing: tone down the bias. Exp Cell Res 2014; 322(1): 12-20.
Walsh PS, Erlich HA, Higuchi R. Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl 1992; 1(4): 241-50.
Chhibber A, Schroeder BG. Single-molecule polymerase chain reaction reduces bias: application to DNA methylation analysis by bisulfite sequencing. Anal Biochem 2008; 377(1): 46-54.
Jordan MR, Kearney M, Palmer S, et al. Comparison of standard PCR/cloning to single genome sequencing for analysis of HIV-1 populations. J Virol Methods 2010; 168(1-2): 114-20.
Gorzer I, Guelly C, Trajanoski S, et al. The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing. J Virol Methods 2010; 169(1): 248-52.
Kanagawa T. Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng 2003; 96(4): 317-23.
Liu J, Song H, Liu D, et al. Extensive recombination due to heteroduplexes generates large amounts of artificial gene fragments during PCR. PLoS One 2014; 9(9): e106658.
Liu SL, Rodrigo AG, Shankarappa R, et al. HIV quasispecies and resampling. Science 1996; 273(5274): 415-6.
Meyerhans A, Vartanian JP, Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res 1990; 18(7): 1687-91.
Malet I, Belnard M, Agut H, et al. From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity. J Virol Methods 2003; 109(2): 161-70.
Ulrich A, Andersen KR, Schwartz TU. Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints. PLoS One 2012; 7(12): e53360.
Quail MA, Otto TD, Gu Y, et al. Optimal enzymes for amplifying sequencing libraries. Nat Methods 2011; 9(1): 10-1.
Aird D, Ross MG, Chen WS, et al. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 2011; 12(2): R18.
Oyola SO, Otto TD, Gu Y, et al. Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes. BMC Genomics 2012; 13: 1.
Liang RH, Mo T, Dong W, et al. Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing. Nucleic Acids Res 2014; 42(12): e98.
Casbon JA, Osborne RJ, Brenner S, et al. A method for counting PCR template molecules with application to next-generation sequencing. Nucleic Acids Res 2011; 39(12): e81.
Fu GK, Hu J, Wang PH, et al. Counting individual DNA molecules by the stochastic attachment of diverse labels. Proc Natl Acad Sci USA 2011; 108(22): 9026-31.
Kearney MF, Spindler J, Wiegand A, et al. Lower pre-ART intra-participant HIV-1 pol diversity may not be associated with virologic failure in adults. PLoS One 2018; 13(1): e0190438.
Kinde I, Wu J, Papadopoulos N, et al. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci USA 2011; 108(23): 9530-5.
Ebbert MT, Wadsworth ME, Staley LA, et al. Evaluating the necessity of PCR duplicate removal from next-generation sequencing data and a comparison of approaches. BMC Bioinformatics 2016; 17(Suppl. 7): 239.
Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/ Map format and SAM tools. Bioinformatics 2009; 25(16): 2078-9.
Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 2011; 27(21): 2987-93.
Palmer S, Wiegand AP, Maldarelli F, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 2003; 41(10): 4531-6.
Fuentes-Pardo AP, Ruzzante DE. Whole-genome sequencing approaches for conservation biology: Advantages, limitations and practical recommendations. Mol Ecol 2017; 26(20): 5369-406.

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Year: 2018
Published on: 15 August, 2018
Page: [98 - 105]
Pages: 8
DOI: 10.2174/1570162X16666180626152252
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