Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability.

Despite recent advances in nucleic acid delivery systems with the success of LNP vehicles, adeno-associated virus (AAV) remains the leading platform for targeted gene delivery due to its low immunogenicity to humans, high transduction efficiency, and range of serotypes with varying tropisms. Depending on the therapeutic goals and serotype used, different production conditions may be more amenable, generating an ever-growing need for rapid yet robust analytical techniques to support the high-quality manufacturing of AAV. A critical bottleneck exists for assessing full capsids where rapid, high-throughput techniques capable of analyzing a range of serotypes are needed. Here, we present a rapid, high-throughput analytical technique, microfluidic electrophoresis, for the assessment of full capsids compatible with AAV1, AAV2, AAV6, AAV8, and AAV9 without the need for assay modifications or optimizations, and AAV5 with some constraints. The method presented in this study uses a mathematical formulation we developed previously with a reference standard to combine the independently obtained capsid protein and single-stranded DNA (ssDNA) profiles to estimate the percentage of full capsids in a sample of unknown concentration. We assessed the ability to use a single serotype (AAV8) as the reference standard regardless of the serotype of the sample being analyzed so long as the melting temperature (Tm) of the capsids is within 12 °C from the Tm of AAV8. Using this method, we are able to characterize samples ±6.1% with an average analytical turnaround time of <5 min/sample, using only 10 µL/sample at a concentration of 2.5 × 1012 VG/mL.

Enzymatic isolation and microfluidic electrophoresis analysis of residual dsRNA impurities in mRNA vaccines and therapeutics.

The versatility, rapid development, and ease of production scalability of mRNA therapeutics have placed them at the forefront of biopharmaceutical research. However, despite their vast potential to treat diseases, their novelty comes with unsolved analytical challenges. A key challenge in ensuring sample purity has been monitoring residual, immunostimulatory dsRNA impurities generated during the in vitro transcription of mRNA. Here, we present a method that combines an enzyme, S1 nuclease, to identify and isolate dsRNA from an mRNA sample with a microfluidic electrophoresis analytical platform to characterize the impurity. After the method was developed and optimized, it was tested with clinically relevant, pseudouridine-modified 700 and 1800 bp dsRNA and 818-4451 nt mRNA samples. While the treatment impacted the magnitude of the fluorescent signal used to analyze the samples due to the interference of the buffer with the labeling of the sample, this signal loss was mitigated by 8.8× via treatment optimization. In addition, despite the mRNA concentration being up to 400× greater than that of the dsRNA, under every condition, there was a complete disappearance of the main mRNA peak. While the mRNA peak was digested, the dsRNA fragments remained physically unaffected by the treatment, with no change to their migration time. Using these samples, we detected 0.25% dsRNA impurities in mRNA samples using 15 µL with an analytical runtime of 1 min per sample after digestion and were able to predict their size within 8% of the expected length. The short runtime, sample consumption, and high throughput compatibility make it suitable to support the purity assessment of mRNA during purification and downstream.

A microfluidic electrophoretic dual dynamic staining method for the identification and relative quantitation of dsRNA contaminants in mRNA vaccines.

mRNA vaccines (i.e., COVID-19 vaccine) offer various advantages over traditional vaccines in preventing and reducing disease and shortening the time between pathogen discovery and vaccine creation. Production of mRNA vaccines results in several nucleic acid and enzymatic by-products, most of which can be detected and removed; however, double-stranded RNA (dsRNA) contaminants pose a particular challenge. Current purification and detection platforms for dsRNA vary in effectiveness, with problems in scalability for mass mRNA vaccine production. Effectively detecting dsRNA is crucial in ensuring the safety and efficacy of the vaccines, as these strands can cause autoimmune reactions with length-symptom dependency and enhance mRNA degradation. We present a new microfluidics method to rapidly identify and quantify dsRNA fragments in mRNA samples. Our innovation exploits the differences in the dynamic staining behavior between mRNA and dsRNA molecules to detect dsRNA contaminants in a high throughput approach. The limit of detection of the system for dsRNA was estimated to be between 17.7-76.6 pg µL-1 with a maximum loading capacity of mRNA of 12.99 ng µL-1. Based on these estimated values, our method allows for the detection of dsRNA contaminants present in percentages as low as 0.14-0.59% compared to the total mRNA concentration. Here, we discuss the molecular mechanism of the dynamic staining behavior of dsRNA and mRNA for two different stains. We believe our method will accelerate the mRNA vaccine development from initial development to quality control workflows.

Insight into Increased Recovery and Simplification of Genomic DNA Extraction Methods from Dried Blood Spots.

There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.

An in-depth analysis of four classes of antidepressants quantification from human serum using LC-MS/MS.

Depression is a growing global crisis, with females at a higher rate of diagnosis than males. While the percentage of patients on prescribed antidepressants have tripled over the last two decades, we are still at a crossroad where the discrepancy lies between finding a drug to suit a patient and monitoring the abundance of it in the body to prevent unwanted side effects. Liquid Chromatography tandem mass spectrometry (LC-MS/MS) has garnered the attention of clinicians as a technique to accurately monitor therapeutic drugs in human serum with high specificity and accuracy. This may be a potential solution, but the challenge persists in the realm of sample preparation, where a method is automatable. We have developed and validated an LC-MS/MS-based assay for simultaneous quantification of 4 different classes of commonly prescribed antidepressants in women that is automated using a JANUS G3 Robotic Liquid Handler. Our method utilizes a simple sample preparation technique, utilizing only 20 µL of a serum sample, to accurately measure Bupropion, Citalopram, Desipramine, Imipramine, Olanzapine, Sertraline and Vilazodone across a range of 1.0 to 230 ng/mL. Our method exhibits a linearity of R2 ≥ 0.99 when detected in MRM mode and % CV of ≤ 20% for all analytes across the board. In addition, we have designed a prototype that can be utilized at a clinical mass spectrometry lab and assessed the long-term use of this prototype using an accelerated stability study. Overall, our developed method has the potential to be translated to clinical settings to monitor postpartum depression for a large number of patient samples using automation.

An Optimized CoBRA Method for the Microfluidic Electrophoresis Detection of Breast Cancer Associated RASSF1 Methylation.

Although breast cancer screening assays exist, many are inaccessible and have high turnaround times, leaving a significant need for better alternatives. Hypermethylation of tumor suppressor genes is a common epigenetic marker of breast cancer. Methylation tends to occur most frequently in the promoter and first exon regions of genes. Preliminary screening tests are crucial for informing patients whether they should pursue more involved testing. We selected RASSF1, previously demonstrated to be aberrantly methylated in liquid biopsies from breast cancer patients, as our gene of interest. Using CoBRA as our method for methylation quantification, we designed unique primer sets that amplify a portion of the CpG island spanning the 5' end of the RASSF1 first exon. We integrated the CoBRA approach with a microfluidics-based electrophoresis quantification system (LabChip) and optimized the assay such that insightful results could be obtained without post-PCR purification or concentration, two steps traditionally included in CoBRA assays. Circumventing these steps resulted in a decreased turnaround time and mitigated the laboratory machinery and reagent requirements. Our streamlined technique has an estimated limit of detection of 9.1 ng/µL of input DNA and was able to quantify methylation with an average error of 4.3%.

Ultrasound frequency sonication facilitates high-throughput and uniform dissociation of cellular aggregates and tissues.

A sample preparation step involving dissociation of tissues into their component cells is often required to conduct analysis of nucleic acids and other constituents from tissue samples. Frequently, the extracellular matrix and cell-cell adhesions are disrupted via treatment with a chemical dissociating reagent or various mechanical forces. In this work, a new, high-throughput, multiplexed method of dissociating tissues and cellular aggregates into single cells using ultrasound frequency bath sonication is explored and characterized. Different operating parameters are evaluated, and a treatment protocol with potential for uniform, high-throughput tissue dissociation is compared to the existing best chemical and orbital plate shaking protocol. Metrics such as percent dissociation, cellular recovery, average aggregate size, proportion of various aggregate sizes, membrane circularity, and cellular viability are subsequently assessed and found to be favorable. In optimized conditions, 53 ±â€¯8% of 1 mm biopsy cores are dissociated within 30 min using sonication alone, surpassing leading high-throughput orbital plate shaking techniques five-fold. Chemical digestion is also 2 times more effective when complexed with sonication rather than orbital plate shaking. RNA content, quality, and expression are found to be superior to the standard protocol in terms of transcriptional preservation.

An investigation into simplifying total RNA extraction with minimal equipment using a low volume, electrokinetically driven microfluidic protocol.

Current methods for total RNA extraction are time-consuming and require several hands-on steps and specialized equipment. Microfluidic devices can offer the opportunity to reduce the number of hands-on steps, decrease the volumes of reagents required for purification, and make extraction high throughput. Here, we investigated the translation of a high volume magnetic bead-based total RNA extraction method (from human whole blood) onto a low input volume microfluidic device. Our results first show that RNA integrity is maintained when the reagent volumes are scaled down by a factor of 22 and the wash buffers are combined 1:1. With our microfluidic method, the number of wash steps can be reduced from four to one. Thus, the time to complete RNA extraction can be reduced from 2 h to 40 min. These manipulations to the conventional protocol yielded RNA amplifiable within 40 cycles of reverse transcription quantitative PCR (RT-qPCR) when using the microfluidic device to simplify the wash steps. To improve the purification of the RNA during the bead transport through the microchannel, we also investigated the effect of a synergetic application of the electrokinetic flow. Our results show that DNase I and other contaminants surrounding the beads get washed away more effectively via electrophoretic transport. Most notably, RNA adsorption on the beads is strong enough to counter electrophoretically-driven desorption. In all, our work opens new ways to extract high-quality total RNA rapidly and simply from a small quantity of blood, making the process of RNA extraction more accessible.

Electro-DBS: A Simple Method to Rapidly Extract Genomic DNA from Dried Blood Spots.

Dried blood spots (DBSs) have been used for more than 50 years and are used as a method of sample collection for pharmacological testing, genetic analyses, monitoring of viral infections, and more. Protocols for nucleic acid extraction from DBSs involve several steps that require specialized equipment and can be lengthy. Thus, we sought to explore ways to reduce the analytical burden of DBSs. We developed a DBS extraction method that uses the synergistic action of electrophoretic and diffusive transport mechanisms to extract genomic DNA (gDNA) through the DBS matrix. This method (which we termed "Electro-DBS") reduces the time of extraction from 40 min to 5 min and removes the need for heat, shaking, and DNA purification steps while maintaining gDNA quality and yield. We found that the electrophoretic transport speeds up gDNA elution, allowing for the diffusive transport of polymerase chain reaction (PCR) inhibitors to be minimized due to a reduced elution time. Overall, this work added mechanistic insight into the extraction of DNA from DBSs and developed a method that was ideal for point of care and automation.

A Comparative Analysis of Deep Learning Models for Automated Cross-Preparation Diagnosis of Multi-Cell Liquid Pap Smear Images.

Routine Pap smears can facilitate early detection of cervical cancer and improve patient outcomes. The objective of this work is to develop an automated, clinically viable deep neural network for the multi-class Bethesda System diagnosis of multi-cell images in Liquid Pap smear samples. 8 deep learning models were trained on a publicly available multi-class SurePath preparation dataset. This included the 5 best-performing transfer learning models, an ensemble, a novel convolutional neural network (CNN), and a CNN + autoencoder (AE). Additionally, each model was tested on a novel ThinPrep Pap dataset to determine model generalizability across different liquid Pap preparation methods with and without Deep CORAL domain adaptation. All models achieved accuracies >90% when classifying SurePath images. The AE CNN model, 99.80% smaller than the average transfer model, maintained an accuracy of 96.54%. During consecutive training attempts, individual transfer models had high variability in performance, whereas the CNN, AE CNN, and ensemble did not. ThinPrep Pap classification accuracies were notably lower but increased with domain adaptation, with ResNet101 achieving the highest accuracy at 92.65%. This indicates a potential area for future improvement: development of a globally relevant model that can function across different slide preparation methods.

Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

Adeno-associated virus (AAV) has shown great potential in gene therapy due to its low immunogenicity, lack of pathogenicity to humans, and ability to provide long-term gene expression in vivo. However, there is currently a need for fast, high-throughput characterization systems that require low volumes for the determination of its sample composition in terms of full and empty capsids since empty capsids are a natural byproduct of AAV synthesis. To address this need, the following study proposes a high-throughput electrophoresis-mediated microfluidics approach that is independent of sample input concentration to estimate the composition of a given sample by combining its protein and ssDNA information relative to a standard. Using this novel approach, we were able to estimate the percentage of full capsids of six AAV8 samples with an average deviation from the actual percentage of 4%. The experiments used for these estimations were conducted with samples of varying percentages of full capsids (21-75%) and varying concentrations (5 × 1011-1 × 1012 VP/mL) with a total volume requirement of 3-10 µL for triplicate analysis of the sample. This method offers a rapid way to evaluate the quality and purity of AAV products. We believe that our method addresses the critical need as recognized by the gene and molecular therapy community.

Electric-field facilitated rapid and efficient dissociation of tissues Into viable single cells.

Single-Cell Analysis is a growing field that endeavors to obtain genetic profiles of individual cells. Disruption of cell-cell junctions and digestion of extracellular matrix in tissues requires tissue-specific mechanical and chemical dissociation protocols. Here, a new approach for dissociating tissues into constituent cells is described. Placing a tissue biopsy core within a liquid-filled cavity and applying an electric field between two parallel plate electrodes facilitates rapid dissociation of complex tissues into single cells. Different solution compositions, electric field strengths, and oscillation frequencies are investigated experimentally and with COMSOL Multiphysics. The method is compared with standard chemical and mechanical approaches for tissue dissociation. Treatment of tissue samples at 100 V/cm 1 kHz facilitated dissociation of 95 ± 4% of biopsy tissue sections in as little as 5 min, threefold faster than conventional chemical-mechanical techniques. The approach affords good dissociation of tissues into single cells while preserving cell viability, morphology, and cell cycle progression, suggesting utility for sample preparation of tissue specimens for direct Single-Cell Analysis.

Sequence to size-based separation using microfluidic electrophoresis for targeted cell-free DNA analysis.

The aim of this research is to present a new method to identify and separate target DNA of the same size, in base pairs (bp), into different sizes based on the targeted sequences. This sequence-specific analysis can then be used to evaluate the presence of multiple targeted analytes in a sample without the need for fluorescence detection. This work displays the feasibility of this method using multiple different 150 bp target sequences separated via microfluidic electrophoresis into 230 bp to 330 bp peaks. Using a combination of denaturation, hybridization, ligation, purification, and universal amplification, this represents a simple, robust method for targeted analysis of short DNA sequences. This work shows a limit of detection of 3 pg (∼1.825 x 107 copies) of input DNA using 20 PCR cycles and the ability for the method to be used for short DNA sequences extracted from a plasma sample, most importantly cell-free DNA. Overall, this method has the potential to be used for mutation detection and multiplexed analysis without the need for multiple fluorophores or significant optimization due to varying melting temperatures between PCR primers and can qualitatively evaluate the presence of specific target sequences in a variety of molecular diagnostic applications.

Pre-eclampsia: a Scoping Review of Risk Factors and Suggestions for Future Research Direction.

Abstract: Most of maternal deaths are preventable, and one-quarter of maternal deaths are due to pre-eclampsia and eclampsia. Prenatal screening is essential for detecting and managing pre-eclampsia. However, pre-eclampsia screening is solely based on maternal risk factors and has low (< 5% in the USA) detection rates. This review looks at pre-eclampsia from engineering, public health, and medical points of view. First, pre-eclampsia is defined clinically, and the biological basis of established risk factors is described. The multiple theories behind pre-eclampsia etiology should serve as the scientific basis behind established risk factors for pre-eclampsia; however, African American race does not have sufficient evidence as a risk factor. We then briefly describe predictive statistical models that have been created to improve screening detection rates, which use a combination of biophysical and biochemical biomarkers, as well as aspects of patient medical history as inputs. Lastly, technologies that aid in advancing pre-eclampsia screening worldwide are explored. The review concludes with suggestions for more robust pre-eclampsia research, which includes diversifying study sites, improving biomarker analytical tools, and for researchers to consider studying patients before they become pregnant to improve pre-eclampsia detection rates. Additionally, researchers must acknowledge the systemic racism involved in using race as a risk factor and include qualitative measures in study designs to capture the effects of racism on patients. Lay Summary: Pre-eclampsia is a pregnancy-specific hypertensive disorder that can affect almost every organ system and complicates 2-8% of pregnancies globally. Here, we focus on the biological basis of the risk factors that have been identified for the condition. African American race currently does not have sufficient evidence as a risk factor and has been poorly studied. Current clinical methods poorly predict a patient's likelihood of developing pre-eclampsia; thus, researchers have made statistical models that are briefly described in this review. Then, low-cost technologies that aid in advancing pre-eclampsia screening are discussed. The review ends with suggestions for research direction to improve pre-eclampsia screening in all settings.Overall, we suggest that the future of pre-eclampsia screening should aim to identify those at risk before they become pregnant. We also suggest that the clinical standard of assessing patient risk solely on patient characteristics needs to be reevaluated, that study locations of pre-eclampsia research need to be expanded beyond a few high-income countries, and that low-cost technologies should be developed to increase access to prenatal screening.

DirectDetect SARS-CoV-2 Direct Real-Time RT-PCR Study Using Patient Samples.

COVID-19 is an infectious disease that caused a global pandemic affecting people worldwide. As disease detection and vaccine rollout continue to progress, there is still a need for efficient diagnostic tools to satisfy continued testing needs. This preliminary study evaluated a novel SARS-CoV-2 diagnostic test called DirectDetect SARS-CoV-2 Direct Real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on a limited sample size of 24 respiratory samples from 14 SARS-CoV-2-positive patients. The test is advantageous compared to others on the market since it does not require viral transport medium or viral RNA extraction prior to nucleic acid amplification and detection. This capability transforms the hours-long sample preparation time into a minutes-long procedure while also eliminating the need for many costly reagents which may be difficult to obtain during the surge in nucleic acid-based testing during the pandemic. The results show a positive agreement of 94.7, 100, and 94.7% between dry sample swabs, treated samples, and untreated samples tested using the DirectDetect SARS-CoV-2 Direct Real-time RT-PCR compared to tests used in a clinical laboratory, respectively. The findings indicate that DirectDetect can be used for multiple different sample types while reducing the number of reagents and time needed for diagnosis. Although this study shows promising results using the DirectDetect results, further validation of this test using a larger sample set is required to assess the true performance of this test.

Simultaneous detection of salivary cortisol and cortisone using an automated high-throughput sample preparation method for LC-MS/MS.

High-Performance Liquid Chromatography-Tandem Mass Spectrometry has emerged triumphant over the years as a reliable and high throughput clinical instrument to assess different metabolic irregularities. One of the latest applications of LC-MS/MS has been in the field of quantification of glucocorticoids, such as cortisol and cortisone from saliva, that can be an indicator of abnormalities such as Congenital Adrenal Hyperplasia (CAH), Cushing's Syndrome, and Addison's disease. We have developed and validated an LC-MS/MS-based assay for simultaneous detection of cortisol and cortisone in human saliva, which requires only 20 µL of sample, to measure cortisol across a 0.5 - 70 ng/mL range, and cortisone across a 1.2- 100 ng/mL range, respectively. The developed method exhibits linearity of R2>0.99, for both analytes, inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 20%. Using dilute-and-shoot for sample preparation, we have exhibited sample accuracy of 100±20 for the assay calibrators, and integrated Needle Wash Solvent Chemistry for minimal sample carryover, making it adaptable for crucial for potential diagnostic use. We have exhibited a method that is simplistic, specific, and highly automatable on liquid handling platforms, such as the JANUS® G3 Workstation. With our innovation, we have introduced a potential to test 81-unknown samples in singlicate within an on-deck plating time of fewer than four minutes. We performed additional verification studies, including an accelerated stability study and a freeze-thaw study showcasing the potential long-term usability of our proposed prototype kit. Overall, this work presents an optimized LC-MS/MS method with automated sample preparation that is ready to be utilized for cortisol-cortisone detection for clinical diagnostic contexts related to Cushing's Disease, among other adrenal and endocrine disorders.

A microfluidic platform for high-purity cell free DNA extraction from plasma for non-invasive prenatal testing.

OBJECTIVES: Increase the yield and purity of cell-free DNA (cfDNA) extracted from plasma for non-invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. METHODS: This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead-based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. RESULTS: When performing the first wash step of this assay on the microfluidic chip with 300 V applied across the channel there was a six-fold increase in the A260 /A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260 /A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. CONCLUSIONS: This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.

A Closer Look into FDA-EUA Approved Diagnostic Techniques of Covid-19.

The 2019 coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 virus, caused a worldwide pandemic in 2020 and is the most urgent health issue worldwide. In this review, we highlight the details of Food and Drug Administration-Emergency Use Authorizations approved diagnostics kits, focusing on the similarities and differences. It is essential to understand the currently available options and the advantages and disadvantages each provides to select the appropriate products that maximize the testing efficiency. We believe this work will provide a holistic evaluation of the current COVID-19 diagnostic resources, including variations across the countries, and guide developing novel diagnostic techniques to improve and optimize the current testing options.

SARS-CoV-2 Variants in Rhode Island.

COVID-19 is a worldwide public health emergency caused by SARS-CoV-2. Genomic surveillance of SARS-CoV-2 emerging variants is important for pandemic monitoring and informing public health responses. Through an interstate academic-public health partnership, we established Rhode Island's capacity to sequence SARS-CoV-2 genomes and created a systematic surveillance program to monitor the prevalence of SARS-CoV-2 variants in the state. We describe circulating SARS-CoV-2 lineages in Rhode Island; provide a timeline for the emerging and expanding contribution of variants of concern (VOC) and variants of interest (VOI), from their first introduction to their eventual predominance over other lineages; and outline the frequent identification of known adaptively beneficial spike protein mutations that appear to have independently arisen in non-VOC/non-VOI lineages. Overall, the described Rhode Island- centric genomic surveillance initiative provides a valuable perspective on SARS-CoV-2 in the state and contributes data of interest for future epidemiological studies and state-to-state comparisons.