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.

Adenoviromics: Mining the Human Adenovirus Species D Genome

“…The National Human Genome Research Institute project to identify functional elements in the human genome (Encyclopedia of DNA Elements, or ENCODE) identified functionality in much of the human genome previously without known utility (Consortium et al, 2007 ; Qu and Fang, 2013 ; Kellis et al, 2014 ) The double-stranded DNA genomes of HAdV also contain regions with no known function. Transcriptional profiling of host gene expression has been studied after HAdV infection (Dorer et al, 2011 ) However, although viral transcriptomes have been reported for several viruses, most notably dengue, varicella zoster, and Epstein-Barr viruses (Ortmann et al, 2008 ; Ertl et al, 2011 ; Nagel et al, 2011 , 2013 ; Arvey et al, 2013 ; Sujayanont et al, 2014 ), a de novo HAdV transcriptome has not been reported. Wu and coworkers used deep RNA sequencing to confirm known bat AdV transcripts (Wu et al, 2013 ), but did not investigate “noncoding” regions.…”

Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA

“…Towards the limit of detection, it was more difficult to distinguish the signal from background for RCA when compared to TN-RCA (Figure S11A,B). This can be explained by incorporation of labelled dNTPs into accessible 3′-ends generated by the normal padlock and start primer and present in denatured HeLa genomic DNA (what is the basis of the whole genome amplification method [54]) and subsequent detection as background.…”

Complete Genome Sequence of West Nile Virus (WNV) from the First Human Case of Neuroinvasive WNV Infection in Cyprus

“…Previously, it was shown that whole-transcriptome amplification techniques, which are based on multiple-displacement amplification, can be successfully used for amplification of RNA virus genomes ( 4 ). Because of the limited amount of viral genomic material available, a similar approach was employed here using the Quantitect whole-transcriptome kit (Qiagen) to increase the amount of cDNA necessary for sequencing the complete genome.…”