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).