Dataset Information

An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

ABSTRACT: The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5'-end of transcripts. Notably 183 - 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5'-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5'-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5'-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5'-UTR introns in An. stephensi.

SUBMITTER: Reid W

PROVIDER: S-EPMC6169391 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Publications

An <i>Anopheles stephensi</i> Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

Reid William W Pilitt Kristina K Alford Robert R Cervantes-Medina Adriana A Yu Hao H Aluvihare Channa C Harrell Rob R O'Brochta David A DA

G3 (Bethesda, Md.) 20181003 10

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, <i>Anopheles stephensi</i> and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the teta ...[more]

PMID: 30135106

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Project description:BackgroundMalaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue.ResultsWe obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi.Conclusion3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

Project description: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.

Dataset Information

An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

Publications

An <i>Anopheles stephensi</i> Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

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