An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster.

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia, Canada. The use of the panel for parentage applications was evaluated using multiple generations of oysters from a breeding program on Vancouver Island, Canada (n = 181) and families selected for Ostreid herpesvirus-1 resistance from the Molluscan Broodstock Program in Oregon, USA (n = 136). Population characterization was evaluated using wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicates showed high genotype concordance (97.5%; n = 68 replicates). Parentage analysis found suspected pedigree and sample handling errors, demonstrating the panel's value for quality control in breeding programs. Suspected null alleles were identified and found to be largely population dependent, suggesting population-specific variation impacting target amplification. Null alleles were identified using existing data without the need for pedigree information, and once they were removed, assignment rates increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided.

An integrated chance constraints approach for optimal vaccination strategies under uncertainty for COVID-19.

Despite concerted efforts by health authorities worldwide to contain COVID-19, the SARS-CoV-2 virus has continued to spread and mutate into new variants with uncertain transmission characteristics. Therefore, there is a need for new data-driven models for determining optimal vaccination strategies that adapt to the new variants with their uncertain transmission characteristics. Motivated by this challenge, we derive an integrated chance constraints stochastic programming (ICC-SP) approach for finding vaccination strategies for epidemics that incorporates population demographics for any region of the world, uncertain disease transmission and vaccine efficacy. An optimal vaccination strategy specifies the proportion of individuals in a given household-type to vaccinate to bring the reproduction number to below one. The ICC-SP approach provides a quantitative method that allows to bound the expected excess of the reproduction number above one by an acceptable amount according to the decision-maker's level of risk. This new methodology involves a multi-community household based epidemiology model that uses census demographics data, vaccination status, age-related heterogeneity in disease susceptibility and infectivity, virus variants, and vaccine efficacy. The new methodology was tested on real data for seven neighboring counties in the United States state of Texas. The results are promising and show, among other findings, that vaccination strategies for controlling an outbreak should prioritize vaccinating certain household sizes as well as age groups with relatively high combined susceptibility and infectivity.

COVID-19 vaccination policies under uncertain transmission characteristics using stochastic programming.

We develop a new stochastic programming methodology for determining optimal vaccination policies for a multi-community heterogeneous population. An optimal policy provides the minimum number of vaccinations required to drive post-vaccination reproduction number to below one at a desired reliability level. To generate a vaccination policy, the new method considers the uncertainty in COVID-19 related parameters such as efficacy of vaccines, age-related variation in susceptibility and infectivity to SARS-CoV-2, distribution of household composition in a community, and variation in human interactions. We report on a computational study of the new methodology on a set of neighboring U.S. counties to generate vaccination policies based on vaccine availability. The results show that to control outbreaks at least a certain percentage of the population should be vaccinated in each community based on pre-determined reliability levels. The study also reveals the vaccine sharing capability of the proposed approach among counties under limited vaccine availability. This work contributes a decision-making tool to aid public health agencies worldwide in the allocation of limited vaccines under uncertainty towards controlling epidemics through vaccinations.

Development of a new AgriSeq 4K mid-density SNP genotyping panel and its utility in pearl millet breeding.

Pearl millet is a crucial nutrient-rich staple food in Asia and Africa and adapted to the climate of semi-arid topics. Since the genomic resources in pearl millet are very limited, we have developed a brand-new mid-density 4K SNP panel and demonstrated its utility in genetic studies. A set of 4K SNPs were mined from 925 whole-genome sequences through a comprehensive in-silico pipeline. Three hundred and seventy-three genetically diverse pearl millet inbreds were genotyped using the newly-developed 4K SNPs through the AgriSeq Targeted Genotyping by Sequencing technology. The 4K SNPs were uniformly distributed across the pearl millet genome and showed considerable polymorphism information content (0.23), genetic diversity (0.29), expected heterozygosity (0.29), and observed heterozygosity (0.03). The SNP panel successfully differentiated the accessions into two major groups, namely B and R lines, through genetic diversity, PCA, and structure models as per their pedigree. The linkage disequilibrium (LD) analysis showed Chr3 had higher LD regions while Chr1 and Chr2 had more low LD regions. The genetic divergence between the B- and R-line populations was 13%, and within the sub-population variability was 87%. In this experiment, we have mined 4K SNPs and optimized the genotyping protocol through AgriSeq technology for routine use, which is cost-effective, fast, and highly reproducible. The newly developed 4K mid-density SNP panel will be useful in genomics and molecular breeding experiments such as assessing the genetic diversity, trait mapping, backcross breeding, and genomic selection in pearl millet.

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology.

The gold standard method for serotyping Escherichia coli has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping E. coli by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins (stx 1, and stx 2) and intimin (eae) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli, the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx 1 , stx 2, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx 1a,c,d (3 of 3 strains), stx 2c-e,g (8 of 8 strains), stx 2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli.