In Silico Karyotyping of Chromosomally Polymorphic Malaria Mosquitoes in the Anopheles gambiae Complex

Abstract

Chromosomal inversion polymorphisms play an important role in adaptation to environmental heterogeneities. For mosquito species in the Anopheles gambiae complex that are significant vectors of human malaria, paracentric inversion polymorphisms are abundant and are associated with ecologically and epidemiologically important phenotypes. Improved understanding of these traits relies on determining mosquito karyotype, which currently depends upon laborious cytogenetic methods whose application is limited both by the requirement for specialized expertise and for properly preserved adult females at specific gonotrophic stages. To overcome this limitation, we developed sets of tag single nucleotide polymorphisms (SNPs) inside inversions whose biallelic genotype is strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural variation. Beginning with principal components analysis (PCA) of population samples, applied to windows of the genome containing individual chromosomal rearrangements, we classified samples into three inversion genotypes, distinguishing homozygous inverted and homozygous uninverted groups by inclusion of the small subset of specimens in Ag1000G that are associated with cytogenetic metadata. We then assessed the correlation between candidate tag SNP genotypes and PCA-based inversion genotypes in our training sets, selecting those candidates with >80% agreement. Our initial tests both in held-back validation samples from Ag1000G and in data independent of Ag1000G suggest that when used for in silico inversion genotyping of sequenced mosquitoes, these tags perform better than traditional cytogenetics, even for specimens where only a small subset of the tag SNPs can be successfully ascertained.

Inversion Genotyping in theAnopheles gambiaeComplex Using High-Throughput Array and Sequencing Platforms

“…Previous work has shown that applying candidate tags to a taxon in which they are not 459 valid has the effect of downwardly biasing the average number of inferred alternate 460 alleles (Love et al 2019). Consistent with this, if 2Rd tags in the present study were 461 inappropriately applied to An.…”

“…Beginning with a ranked list of tags based on descending genotypic concordance, 212 we applied filters (described below) that narrowed the numbers of tags based on design 213 criteria unique to each platform. To ensure that we retained at least six tag SNPs per inversion for genotyping, we were 235 compelled to reduce the genotypic concordance threshold below the 0.8 level imposed 236 in Love et al (2019). Even lowering the threshold to 0.7 for 2Rc tags in An.…”

“…Studies employing this tool in the laboratory suggested that the inverted orientation was 105 associated with increased thermal and desiccation tolerance, a thicker cuticle, higher 106 body water content, a more aggressive upregulation of heat-responsive genes such as 107 heat shock genes, and a higher energy budget relative to the alternative (2L+ a ) 108 inside the rearranged region whose allelic state is strongly correlated with inversion 131 orientation regardless of their position relative to the breakpoints (Love et al 2019). To 132 our knowledge, no DNA-based molecular assays exist for the genotyping of the other 133 inversions in An.…”

“…coluzzii 136 database of natural variation (Ag1000G; www.malariagen.net/projects/ag1000g) (Miles 137 et al 2017) to identify tag SNPs predictive of inversion orientation for all six common 138 inversion polymorphisms in these species. Using these tags, we developed an 139 algorithm capable of in silico inversion genotyping based on SNPs called from whole 140 genome resequencing data (Love et al 2019). This is a rapid and powerful approach 141 assuming that whole genome sequence data are already available or will be produced 142 for other reasons.…”

“…The tags were ascertained in the 446 Ag1000G variation database, whose content was heavily biased toward An. gambiae at 447 the time of their discovery (Love et al 2019). Available samples of An.…”