Biology subjects

Wilson, M. M.

Publications and source records attributed to Wilson, M. M..

6 recordsLinked to original sources

bioRxiv2023

Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein

The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.

genomics↗

bioRxiv2020

An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis

The Epithelial-Mesenchymal Transition (EMT) and primary ciliogenesis induce stem cell properties in basal Mammary Stem Cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis at intermediate EMT transition states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote BBS11-dependent ubiquitination and inactivation of a central signaling node, GLIS2. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the Mammary-Tumor-initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC biology, mammary gland development, and claudin-low breast cancer formation.

cell biology↗

bioRxiv2019

Detection and discrimination of influenza B Victoria lineage deletion variant viruses by real-time RT-PCR

Influenza B viruses have two genetically and antigenically distinct lineages, B/Victoria/2/1987-like (VIC) and B/Yamagata/16/1988-like (YAM) viruses, that emerged in the 1980s and co-circulate annually during the influenza season. During the 2016-2017 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (AA) deletions in the hemagglutinin protein. Hemagglutination inhibition assays demonstrate that these deletion variant influenza B/VIC viruses are antigenically distinct from each other and from the progenitor B/VIC virus that lacks the deletion. Therefore, there are currently four antigenically distinct HA proteins expressed by influenza B co-circulating: B/YAM, B/VIC V1A (no deletion), B/VIC V1A.1 (two-AA deletion), and B/VIC V1A.2 and V1A.3 (three-AA deletion). The prevalence of these viruses differs across geographic regions, making it critical to have a sensitive, rapid diagnostic assay(s) that detect and distinguish these Influenza B variant viruses during surveillance. Here, we present a real time RT-PCR assay that targets the influenza B/VIC deletion region in the HA gene and detects and distinguishes the influenza B/VIC V1A, B/VIC V1A.1, B/VIC V1A.2 and B/VIC V1A.3 variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost. Coupling this assay with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterization of circulating influenza B viruses. Having accurate and detailed surveillance information on these distinct Influenza B variant viruses will provide insight into the prevalence and geographic distribution and could aid in vaccine recommendations.

molecular biology↗

bioRxiv2019

Phylogenetic synecdoche demonstrates optimality of subsampling and improves recovery of the Blaberoidea phylogeny

Phylogenomics seeks to use next-generation data to robustly infer an organisms evolutionary history. Yet, the practical caveats of phylogenomics motivates investigation of improved efficiency, particularly when quality of phylogenies are questionable. To achieve improvements, one goal is to maintain or enhance the quality of phylogenetic inference while severely reducing dataset size. We approach this goal by designing an optimized subsample of data with an experimental design whose results are determined on the basis of phylogenetic synecdoche - a comparison of phylogenies inferred from a subsample to phylogenies inferred from the entire dataset. We examine locus mutation rate, saturation, evolutionary divergence, rate heterogeneity, selection, and a priori information content as traits that may determine optimality. Our controlled experimental design is based on 265 loci for 102 blaberoidean cockroaches and 22 outgroup species. High phylogenetic utility is demonstrated by loci with high mutation rate, low saturation, low sequence distance, low rate heterogeneity, and low selection. We found that some phylogenetic information content estimators may not be meaningful for assessing information content a priori. We use these findings to design concatenated datasets with an optimized subsample of 100 loci. The tree inferred from the optimized subsample alignment was largely identical to that inferred from all 265 loci but with less evidence of long branch attraction and improved statistical support. In sum, optimized subsampling can improve tree quality while reducing data collection costs and yielding 4-6x improvements to computation time in tree inference and bootstrapping.

evolutionary biology↗

bioRxiv2019

Mitigating Pandemic Risk with Influenza A Virus Field Surveillance at a Swine-Human Interface

Working overnight at a large swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, nanopore-sequenced 13 IAV genomes from samples collected, and in real-time, determined that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current pre-pandemic candidate vaccine viruses (CVV). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 hours after unpacking the mobile lab. Swine are important animal IAV reservoirs that have given rise to pandemic viruses via zoonotic transmission. Genomic analyses of IAV in swine are critical to understanding pandemic risk of viruses in this reservoir, and characterization of viruses circulating in exhibition swine enables rapid comparison to current seasonal influenza vaccines and CVVs. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes: A(H1N1), A(H3N2) and A(H1N2). Additional analysis of the HA protein sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 portion of the hemagglutinin of these viruses and the most closely related pre-2009 CVV. All virus sequences were emailed to colleagues at CDC who initiated development of a synthetically derived CVV designed to provide an optimal antigenic match with the viruses detected in the exhibition. In subsequent months, this virus caused 13 infections in humans, and was the dominant variant virus in the US detected in 2018. Had this virus caused a severe outbreak or pandemic, our proactive surveillance efforts and CVV derivation would have provided an approximate 8 week time advantage for vaccine manufacturing. This is the first report of the use of field-derived nanopore sequencing data to initiate a real-time, actionable public health countermeasure.

microbiology↗

bioRxiv2018

Complete genome direct RNA sequencing of influenza A virus

For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

genomics↗