Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia

Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew

Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).