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Rivers, A. R.

Publications and source records attributed to Rivers, A. R..

6 recordsLinked to original sources

TAMIPAMI: Software and methods for PAM/TAM identification for CRISPR and OMEGA gene editing systems

Protospacer adjacent motifs (PAMs) and target-adjacent motifs (TAMs) are essential for target recognition by CRISPR-Cas and TnpB nucleases. Here we present TAMIPAMI, an efficient experimental and computational framework for rapid PAM/TAM identification. TAMIPAMI requires only a single control library and Cas or TnpB-treated library, simplifying experimental design, reducing cost, and providing greater accessibility for users. The platform interprets sequencing data with interactive visualizations and introduces a novel algorithm that determines the minimal exact set of degenerate IUPAC sequences describing the observed PAM/TAM patterns. Using this approach, we accurately recovered canonical motifs for several nucleases, including SpCas9, LbCas12a, AsCas12a, BrCas12b, Cas12i1, and AmaTnpB. TAMIPAMI is available as both a web application and command-line tool, ultimately providing an accessible and efficient platform for PAM/TAM discovery and characterization across CRISPR and OMEGA systems.

bioinformatics↗

An Arrayed Transposon Library of Ruegeria pomeroyi DSS-3

The ability to construct defined genetic mutations in many bacteria is difficult and limited. Transposon mutagenesis is often highly efficient, but is not site specific, thus selections are often needed to identify mutants of interest. The construction of arrayed mutant libraries would help to fill this need, though these libraries are costly and time consuming. To enable easier construction of arrayed libraries we developed a workflow and methodology using a hierarchical barcoding scheme to identify mutants within a multiwell plate. We applied this method to the marine Alphaproteobacterium Ruegeria pomeroyi DSS-3 and created a library with over 2,800 disrupted genes.

microbiology↗

Graph-based pangenomics maximizes genotyping density and reveals structural impacts on fungal resistance

The genomic sequences segregating in experimental populations are often highly divergent from the community reference and from one another. Such divergence is problematic under various short-read-based genotyping strategies. In addition, large structural differences are often invisible despite being strong candidates for causal variation. These issues are exacerbated in specialty crop breeding programs with fewer, lower-quality sequence resources. We examined the benefits of complete genomic information, based on long-read assemblies, in a biparental mapping experiment segregating at numerous disease resistance loci in the non-model crop, melon (Cucumis melo). We find that a graph-based approach, which uses both parental genomes, results in 19% more variants callable across the population and raw allele calls with a 2 to 3-fold error-rate reduction, even relative to single reference approaches using a parent genome. We show that structural variation has played a substantial role in shaping two Fusarium wilt resistance loci with known causal genes. We also report on the genetics of powdery mildew resistance, where copy number variation and local recombination suppression are directly interpretable via parental genome alignments. Benefits observed, even in this low-resolution biparental experiment, will inevitably be amplified in more complex populations.

genetics↗

Intrinsic variation in the vertically transmitted insect-specific core virome of Aedes aegypti

Since 2009, local outbreaks of dengue (serotypes 1-3) mediated by Aedes aegypti mosquitoes have occurred in the United States, particularly in Florida (FL). In 2016 and 2017, dengue virus serotype 4 was found alongside several insect-specific viruses (ISVs) in pools of A. aegypti from sites in Manatee County, FL, in the absence of an index case. Although ISVs have been characterized in A. aegypti globally, the constitution of a core virome in natural populations remains unclear. Using mosquitoes sampled from the same area in 2018, we compared baseline ovary viromes of field (G0) and lab (Orlando) A. aegypti via metagenomic RNA sequencing. Across all samples, virome composition varied by sample type (field- or colony-derived). Four ISVs comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a previously described toti-like virus (Totiviridae), unclassified Riboviria, and four previously described orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the toti-like virus, partiti-like virus, and one orthomyxo-like virus were assembled and analyzed phylogenetically. Multigenerational maintenance of these ISVs was confirmed orthogonally by RT-PCR in G0 and G7 mosquitoes, indicating vertical transmission as the mechanism for ISV sustentation. This study provides fundamental information regarding ISV ecology, persistence, and variation in A. aegypti in nature.

microbiology↗

GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes

BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens. FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes. ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

bioinformatics↗

Under-the-radar dengue virus infections in natural populations of Aedes aegypti mosquitoes

The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort on the dengue mosquito vector, Aedes aegypti, and its microbiome, which has been shown to influence virus transmission success. We examined the metavirome of four populations of Ae. aegypti mosquitoes collected in 2016-2017 from Manatee County, Florida. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a two-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, as well as explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of these DENV4 infections in Ae. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak. ImportanceSince 1999, dengue outbreaks in the continental United States (U.S.) involving local transmission have occurred episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from endemic countries. To date, the U.S. public health response to dengue outbreaks is largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of non-outbreak DENV4 transmission and maintenance within a local mosquito vector population in the continental U.S.in the absence of a human case during a two-year time period. Our data suggest that molecular surveillance of mosquito populations in high-risk, high tourism areas of the U.S., may allow for proactive, targeted vector control before potential arbovirus outbreaks.

ecology↗