Chikungunya virus metagenomic sequencing and whole-genome assembly from plasma of infected human blood donors from Puerto Rico
Unbiased metagenomic next-generation sequencing (NGS) was then used as a pan-pathogen screen and to recover the viral genome from the three CHIK-positive samples.  NGS libraries were constructed using the Nextera XT kit (Illumina) and validated as previously described (Nacacche, et al., 2014, Genome Research) followed by 150 base-pair (bp), paired-end sequencing (Illumina MiSeq).  Raw NGS data (3.2 to 32.4 million reads per sample) was analyzed for reads corresponding to known pathogens using the sequence-based ultra-rapid pathogen identification (SURPI) computational pipeline (Nacacche, et al., 2014, Genome Research).  After computational subtraction of human host sequences, alignment was performed against those in the National Center for Biotechnology Information (NCBI) nucleotide (NT) database; the best viral species was selected based on percent mapped read coverage and pairwise identity.  A Caribbean strain from the British Virgin Islands (KJ451624) was identified as the closest matching genome in the NCBI NT database; 95-100% genome coverage was obtained for the three CHIKV-positive donors.  Phylogenetic analysis of the three Puerto Rico CHIKV genomes, together with all 188 publicly available sequenced CHIKV genomes in the reference database, placed the Puerto Rico strains in the Caribbean clade. Molecular clock analysis revealed that this clade, an offshoot of the Southeast Asian/Pacific lineage, was predicted to have recently emerged in the Western Hemisphere in early 2013, with the three Puerto Rico viruses diverging from the other Caribbean strains 1.7 years ago.