Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and High-throughput Sequencing
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RESEARCH
Open Access
Analysis of Quasispecies of Avain Leukosis Virus Subgroup J Using Sanger and Highthroughput Sequencing Fanfeng Meng1†, Xuan Dong1†, Tao Hu2, Yingnan Liu1, Yingjie Zhao1, Yanyan Lv1, Shuang Chang1, Peng Zhao1* and Zhizhong Cui1*
Abstract Background: Avian leukosis viruses subgroup J (ALV-J) exists as a complex mixture of different, but closely related genomes named quasispecies subjected to continuous change according to the Principles of Darwinian evolution. Method: The present study seeks to compare conventional Sanger sequencing with deep sequencing using MiSeq platform to study quasispecies dynamics of ALV-J. Results: The accuracy and reproducibility of MiSeq sequencing was determined better than Sanger sequencing by running each experiment in duplicate. According to the mutational rate of single position and the ability to distinguish dominant quasispecies with two sequencing methods, conventional Sanger sequencing technique displayed high randomness due to few sequencing samples, while deep sequencing could reflect the composition of the quasispecies more accurately. In the mean time, the research of quasispecies via Sanger sequencing was simulated and analyzed with the aid of re-sampling strategy with replacement for 1000 times repeat from high-throughput sequencing data, which indicated that the higher antibody titer, the higher sequence entropy, the harder analyzing with the conventional Sanger sequencing, resulted in lower ratios of dominant variants. Conclusions: In sum, deep sequencing is better suited for detecting rare variants comprehensively. The simulation of Sanger sequencing that we propose here will also help to standardize quasispecies researching under different selection pressure based on next-generation sequencing data. Keywords: Quasispecies-Sanger sequencing-deep sequencing-simulation
Background Avian leukosis virus subgroup J (ALV-J), the same as human immunodeficiency virus, displays extensive genetic diversity, reflecting the error prone characteristics of reverse transcriptase-dependent replication, increased recombination rate and continuous selection of more fit viral variants within fluctuating host ecosystems [1]. Therefore, a complex mixture of different, but closely related genomes named quasispecies subjected to continuous change following Principles of Darwinian Evolution such as genetic variation, competition, selection, * Correspondence: [email protected]; [email protected] Fanfeng Meng and Xuan Dong are co-author for this paper. † Equal contributors 1 College of Veterinary Medicine, Shandong Agricultural University, Taian 271018, China Full list of author information is available at the end of the article
interaction and cooperation were formed [2–7]. Viral variations, the primary cause of quasispecies phenomena, are strongly associated with quasispecies transitions, which affect the clinical manifestations of a patient and the antiviral therapeutic response [8, 9]. ALV-J was first isolated in 1988 from meat-type chickens in Britain [10], which sp
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