A Near-Chromosome Level Genome Assembly of Anopheles stephensi

Abstract

Malaria remains a major healthcare risk to growing economies like India, and a chromosome-level reference genome of Anopheles stephensi is critical for successful vector management and understanding of vector evolution using comparative genomics. We report chromosome-level assemblies of an Indian strain, STE2, and a Pakistani strain SDA-500 by combining draft genomes of the two strains using a homology-based iterative approach. The resulting assembly IndV3/PakV3 with L50 of 9/12 and N50 6.3/6.9 Mb had scaffolds long enough for building 90% of the euchromatic regions of the three chromosomes, IndV3s/PakV3s, using low-resolution physical markers and enabled the generation of the next version of genome assemblies, IndV4/PakV4, using HiC data. We have validated these assemblies using contact maps against publicly available HiC raw data from two strains including STE2 and another lab strain of An. stephensi from UCI and compare the quality of the assemblies with other assemblies made available as preprints since the submission of the manuscript. We show that the IndV3s and IndV4 assemblies are sensitive in identifying a homozygous 2Rb inversion in the UCI strain and a 2Rb polymorphism in the STE2 strain. Multiple tandem copies of CYP6a14, 4c1, and 4c21 genes, implicated in insecticide resistance, lie within this inversion locus. Comparison of assembled genomes suggests a variation of 1 in 81 positions between the UCI and STE2 lab strains, 1 in 82 between SDA-500 and UCI strain, and 1 in 113 between SDA-500 and STE2 strains of An. stephensi , which are closer than 1 in 68 variations among individuals from two other lab strains sequenced and reported here. Based on the developmental transcriptome and orthology of all the 54 olfactory receptors (ORs) to those of other Anopheles species, we identify an OR with the potential for host recognition in the genus Anopheles . A comparative analysis of An. stephensi genomes with the completed genomes of a few other Anopheles species suggests limited inter-chromosomal gene flow and loss of synteny within chromosomal arms even among the closely related species.

Characterization of Anopheles stephensi Odorant Receptor 8, an Abundant Component of the Mouthpart Chemosensory Transcriptome

“…A recent genome reassembly of An. stephensi reported 54 Ors [ 42 ], in contrast to the 59 apparent Ors that we identified in the current study. A thorough re-annotation of chemoreceptors, and the OBPs particularly, may be warranted.…”

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly

“…The new An. stephensi assembly not only achieves significant improvements over the existing draft assembly (1044-fold and 56-fold increase in contig N50 and scaffold N50, respectively, and a 97% reduction in assembly gaps), but it is also more contiguous and complete than the latest versions of published assemblies of Anopheles species (single copy BUSCOs 93.7–98.9%) [ 7 , 18 , 19 ], including the extensively studied African malaria vector, An. gambiae (AgamP4) genome (BUSCO 97.4%, contig N50 = 85 kb, scaffold N50 = 49 Mb), arguably the best characterized genome among malaria vectors (Fig.…”

“…Despite being a pioneering model for transgenics and CRISPR gene drive in malaria vectors [ 13 , 17 ], the community studying An. stephensi still relies on draft genome assemblies that do not achieve the completeness and contiguity of reference-grade genomes to reveal all the hidden genetic features [ 7 , 18 , 19 ]. In particular, this limitation obscures genes and repetitive genetic elements that are potentially relevant for understanding parasite transmission or for managing vector populations [ 20 ].…”

The genome trilogy of Anopheles stephensi, an urban malaria vector, reveals structure of a locus associated with adaptation to environmental heterogeneity

“…These gff files were used for gene liftover between UCI and IndCh. This procedure was adapted from our recent publication [3].…”

“…stephensi have been reported recently, including that of a laboratory strain of An. stephensi , STE2, MRA-128, originally collected from Delhi by the NIMR and subsequently maintained at the Walter Reed Hospital[3]. The STE2 strain has been extensively studied, leading to several publications including the complete physical map[4] and a chromosomal study of several inversions via photomaps of loop formation, including the heterozygous 2R b inversion[5].…”