title: Comparison of phi29-based whole genome amplification and whole transcriptome amplification in dengue virus.
abstract: Dengue virus is responsible for 50-100 million new infections annually worldwide. The virus uses error-prone RNA polymerase during genome replication in a host, resulting in the formation of closely related viruses known as quasispecies. The availability of next-generation sequencing technology provides opportunities to analyze viral quasispecies. Before analysis, it is crucial to increase the amount of DNA because of the limited amounts of viral genomic material that can be isolated from a patient. However, using specific primers may overlook the occurrence of possible variations at primer binding sites. To address this problem, the performance of two sequence-independent amplification methods was compared for whole genome amplification (WGA): phi29 DNA polymerase-based WGA and whole transcriptome amplification (WTA). Both methods have the ability to provide complete coverage of the dengue genome from template amounts as low as 1 ng. However, WTA showed greater efficiency in terms of yield (WTA: ~10 μg; phi29-based WGA: ~500 ng) and lower amplification bias. In conclusion, the WTA amplification kit was shown to perform substantially better than phi29 DNA polymerase-based WGA in terms of both final concentration and amplification bias in amplifying small genomes, such as that of the dengue virus.
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Division of Bioinformatics and Data Management for Research, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Center for Emerging and Neglected Infectious Diseases, Mahidol University, Bangkok, Thailand. Electronic address: patcharawan.su@yahoo.com.
authors.forename: Patcharawan
authors.initials: P
authors.lastname: Sujayanont
authors.forename: Kwanrutai
authors.initials: K
authors.lastname: Chininmanu
authors.forename: Boonrat
authors.initials: B
authors.lastname: Tassaneetrithep
authors.forename: Nattaya
authors.initials: N
authors.lastname: Tangthawornchaikul
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
authors.forename: Prapat
authors.initials: P
authors.lastname: Suriyaphol
mesh_terms: D003716:Dengue Virus / Q000235:genetics*; D016679:Genome, Viral; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D059467:Transcriptome; D014773:Virology / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: Amplification bias; Dengue; WGA; WTA; Whole genome amplification; Whole transcriptome amplification; phi29; whole genome amplification; whole transcriptome amplification