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

Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and