Computational protocol: Genetic characterization of measles virus in the Philippines, 2008–2011

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Protocol publication

[…] PCR products were purified using SUPRICTM-PCR (TaKaRa, Otsu, Japan) kit. Direct sequencing of PCR product was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing products were analyzed using the ABI Genetic Analyzer 3130 (Applied Biosystems, Foster City, CA, USA).Phylogenetic analysis was performed using the neighbor-joining (NJ) method in Molecular Evolution Genetic Analysis (MEGA) software 5.0 []. Statistical support was tested using the bootstrap method with 1,000 replicates and values >60% were indicated on the branches. Sequences of reference strains for each genotype and sequences from other countries were downloaded from GenBank and were included in the phylogenetic analysis. Pairwise p-distance for each measles sequence was calculated using MEGA software 5.0.To estimate the rate of evolution and the time to the most recent common ancestor (tMRCA) of MeV in the Philippines, a Bayesian Markov chain Monte Carlo (MCMC) method was used as implemented in the BEAST package 1.8.0 [, ]. For tMRCA analysis, only samples from the Philippines were included in the dataset. Sample collection dates were incorporated in the N gene sequence dataset to calibrate the molecular clock []. The best-fitting nucleotide substitution model was determined using the MEGA software 5.0 []. Based on the smallest Akaike information criterion (AIC) value, the Hasegawa–Kishino–Yano (HKY) model was selected as the best-fitting model []. The dataset was analyzed in BEAST using two different clock models (strict and uncorrelated lognormal relaxed) and two different demographic models (constant and exponential population growth) []. Results were compared to ensure that convergence was attained in each run and statistical summaries were combined after removing 10% as burn-in using the Tracer software in the BEAST package []. The best-fitting model for the dataset was determined by computing the Bayes factors using the Tracer software. Statistical uncertainties in the estimates were indicated by the 95% highest posterior density (HPD) intervals. Maximum clade credibility (MCC) trees were inferred also using the same N and H datasets in the NJ method for comparison of clustering of the Philippine samples with reference strains. Posterior probability values were indicated on the node of MCC trees. To estimate the genetic diversity of MeV overtime, a coalescent-based Bayesian skyline analysis was performed using the identified best-fitting clock and demographic models. Sequences described in this study were submitted to GenBank (N gene: KM066693-KM066717; H gene: KM 066718-KM066816). […]

Pipeline specifications

Software tools MEGA, BEAST
Application Phylogenetics
Organisms Measles morbillivirus