2. Amplification and sequencing of the 4.3kb structural variant insertion site in Uganda and Cameroon populations.
The entire intergenic region between CYP6P5 and CYP6P9bwas amplified, cloned and sequenced to confirm the presence of this structural variant in Uganda and Cameroon An. funestus s.s. populations. PCR amplicons of approximately 1kb (no insertion) and 5.4kb (with the insertion) were cloned into pJET1.2/blunt cloning vectors and Sanger-sequenced. To sequence the full fragment, four additional sequencing primers were used (Table S1). Sequence reads were aligned to reconstruct the full 5.4 kb region. To improve the sequence, pooled template WGS data were aligned to the draft 5.4 kb intergenic sequence flanked by the two coding genes (accession number : OR000399). Results revealed a 4311bp fragment inserted 494bp downstream of the translation stop codon of CYP6P5 and 494bp upstream of the translation start codon of CYP6P9b (Figure 1A & C). Figure 1C shows the deeper coverage across the middle portion of the sequence, suggesting that it is a genomic fragment occurring in multiple copies in the genome.
Analysis of the entire 5.4kb for the open reading frames revealed the presence of 2 open reading frames corresponding to putative transposable elements. BLASTp identified these transposons to match a gag-like protein from Culex pipens (AAB86424.1) and a reverse transcriptase-like protein from Aedes aegypti (AAA29354.1) (Figure 1C).