Biology subjects

Miller, A. L.

Publications and source records attributed to Miller, A. L..

17 recordsLinked to original sources

bioRxiv2026

Calcium and actomyosin pulses drive tricellular zipping to remodel epithelial cell-cell junctions and facilitate cell packing

Epithelial morphogenesis requires coordinated changes in cell geometry, neighbor cell arrangement, and cell packing to generate functional tissue architectures. However, the spatiotemporal regulation of upstream signals that drive local junctional remodeling in highly proliferative developing epithelial tissues remains unclear. Here, we characterize a new junction remodeling process, which we call "tricellular zipping". It resolves long, thin cell extensions generated by cell-division forces that terminate at transient higher-order vertices. We show that tricellular zipping facilitates epithelial cell packing in blastula-stage Xenopus laevis embryos through asymmetric displacement of a tricellular vertex and formation of a de novo bicellular interface. Similarly, we observe tricellular zipping in developing mouse embryos. Combining modeling and live imaging approaches, we find that displacement of the remodeling vertex depends on actomyosin-dependent asymmetric force generation and on enrichment of adaptor proteins coupling actomyosin to junctions, which enables effective force transmission. In contrast, the stationary vertex remains close to mechanical equilibrium and therefore undergoes no net displacement. Furthermore, we find that calcium flashes drive the localized, pulsatile actomyosin contractility required for efficient tricellular zipping. Extending our observations, we show that disrupting transient calcium signaling also affects cell packing in Drosophila embryos, thereby impairing mesoderm internalization. Overall, by combining multidisciplinary and multisystem approaches, we uncover a new mode of junctional remodeling and highlight the importance of spatiotemporally regulated calcium signaling in driving local junctional rearrangements to facilitate morphogenesis.

cell biology↗

bioRxiv2026

NEPTUNE: a neuronal cell death pathway prediction tool used to decipher neurodegeneration using single cell RNA-seq datasets

In the constantly evolving landscape of cell death mechanisms, much of the literature is dominated by research performed in cancer cells and regenerating cell types. However, neurons differ in that they are non-regenerating and post-mitotic. Therefore, assessing neuronal specific disease biomarkers, including cell death markers, without the bias of non-neuronal or cancerous cells, could provide a more biologically accurate neurodegeneration profile. In this study, we developed the Neuronal cEll death Prediction Tool for Understanding Neurodegenerative Events (NEPTUNE), a user-friendly tool for researchers that bioinformatically identifies neuronal cell death markers from user provided differentially expressed gene lists obtained by single cell RNA sequencing data. We applied NEPTUNE to a publicly available single cell RNA sequencing dataset and analysed the progression of photoreceptor degeneration in the rd1 mouse model of severe retinitis pigmentosa. The analysis interpreted the dataset in a neuronal-specific manner and showed that late-stage photoreceptor death is not purely apoptotic but possibly engages overlapping cell death programmes. From the analysis, the user is provided with publishable graphs and a list of cell death markers with fold change, type of impact, cell death type and a strength score. Overall, our tool provides markers specific to neurodegeneration, allows mapping to known WikiPathways and KEGG pathways, performs GO and KEGG enrichment analysis and plots the data for further analysis, greatly facilitating the study of neuron specific degeneration. The tool can be found at: https://neptune.rgtcarvalholab.com/.

bioinformatics↗

bioRxiv2026

Zyxin and LPP differ in their sensing of strained actin filaments at tricellular junctions

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

cell biology↗

bioRxiv2025

Pre-clinical validation of a novel AAV-mediated gene therapy for KCNV2 retinopathy improves visual function in a mouse model and expression in patient organoids

Voltage-gated (Kv) potassium channels are critical for neuronal physiology, and their dysfunction can lead to serious consequences. For example, mutations in the silent modulatory Kv8.2 subunit are known to cause irreversible inherited blindness (KCNV2 retinopathy). This is a currently incurable condition that causes lifelong visual loss, reduced visual acuity, photoaversion, night blindness and abnormal colour vision, alongside a distinctive supernormal electrophysiological (ERG) retinal response to light. In this study, we demonstrate that AAV-mediated gene replacement therapy delivering a codon-optimised human KCNV2 gene subretinally into Kv8.2 knock-out mice significantly restores retinal function. Treated mice exhibited improved ERG responses and correct expression of KCNV2 and its encoded Kv8.2 protein in photoreceptors. Recovery of visually guided scotopic and photopic optomotor responses to wildtype levels was achieved at lower vector doses, highlighting dose-dependent efficacy. Furthermore, treatment of human retinal organoids derived from a KCNV2 patient iPSC line resulted in substantial Kv8.2 protein rescue. This work provides the first preclinical proof-of-concept for the safety and therapeutic potential of gene therapy for KCNV2 retinopathy, laying a strong foundation for future clinical trials.

neuroscience↗

bioRxiv2024

Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling

To preserve barrier function, cell-cell junctions must dynamically remodel during cell shape changes. We have previously described a rapid tight junction repair pathway characterized by local, transient activation of RhoA, termed Rho flares, which repair leaks in tight junctions via promoting local actomyosin-mediated junction remodeling. In this pathway, junction elongation is a mechanical trigger that initiates RhoA activation through an influx of intracellular calcium and recruitment of p115RhoGEF. However, mechanisms that tune the level of RhoA activation and Myosin II contractility during the process remain uncharacterized. Here, we show that the scaffolding protein Anillin localizes to Rho flares and regulates RhoA activity and actomyosin contraction at flares. Knocking down Anillin results in Rho flares with increased intensity but shorter duration. These changes in active RhoA dynamics weaken downstream F-actin and Myosin II accumulation at the site of Rho flares, resulting in decreased junction contraction. Consequently, tight junction breaks are not reinforced following Rho flares. We show that Anillin-driven RhoA regulation is necessary for successfully repairing tight junction leaks and protecting junctions from repeated barrier damage. Together, these results uncover a novel regulatory role for Anillin during tight junction repair and barrier function maintenance. Significance StatementO_LIBarrier function is critical for epithelial tissues. Epithelial cells maintain barrier function via tight junctions, which must be remodeled to allow for cell- and tissue-scale shape changes. How barrier function is maintained and remodeled as epithelial cells change shape remains unclear. C_LIO_LIThe scaffolding protein Anillin is required for generating effective actomyosin contraction to reinforce damaged tight junctions; lack of reinforcement leads to repeated barrier leaks. C_LIO_LIThese findings highlight a novel role for Anillin in tight junction remodeling and suggest that Anillins ability to tune the level and duration of local Rho activation affects the contractile output. C_LI

cell biology↗

bioRxiv2023

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions

Apical cell-cell junctions, including adherens junctions (AJs) and tight junctions (TJs), adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions (BCJs) and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing gastrula-stage Xenopus laevis embryos as a model system, we describe a mechanism by which Vinculin, a mechanosensitive protein, anchors the actomyosin network at TCJs, thus maintaining TJ stability and barrier function. Using an optogenetic approach, we found that acutely increasing junctional tension results in robust recruitment of Vinculin to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that loss of Vinculin results in reduced Actin stability at TCJs. Vinculin knockdown also destabilizes Angulin-1, a key protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJs cannot maintain their proper morphology. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. HighlightsO_LIVinculin is mechanosensitively recruited to tricellular junctions C_LIO_LIVinculins actin-binding function is needed for tricellular actomyosin organization C_LIO_LITricellular tight junctions are unstable when Vinculin is knocked down C_LIO_LIVinculin is required to maintain barrier function at tricellular junctions C_LI

cell biology↗

bioRxiv2023

Neighbor cells restrain furrowing during epithelial cytokinesis

Cytokinesis challenges epithelial tissue homeostasis by generating forces that pull on neighboring cells via cell-cell junctions. Previous work has shown that junction reinforcement at the furrow in Xenopus laevis epithelia regulates the speed of furrowing1. This suggests the cytokinetic array that drives cell division is subject to resistive forces from epithelial neighbor cells. We show here that contractility factors accumulate in neighboring cells near the furrow during cytokinesis. Additionally, increasing neighbor cell stiffness, via [a]-actinin overexpression, or contractility, through optogenetic Rho activation in one neighbor cell, slows or asymmetrically pauses furrowing, respectively. Notably, optogenetic stimulation of neighbor cell contractility on both sides of the furrow induces cytokinetic failure and binucleation. We conclude that forces from the cytokinetic array in the dividing cell are carefully balanced with restraining forces generated by neighbor cells, and neighbor cell mechanics regulate the speed and success of cytokinesis. HighlightsO_LINeighboring cells assemble actomyosin arrays adjacent to the cytokinetic furrow C_LIO_LIOverexpression of an F-actin cross-linker in neighbor cells slows furrowing C_LIO_LIOptogenetic activation of contractility in one neighbor pauses furrow ingression C_LIO_LIHyper-contractility in both neighbors restrains furrowing & cells fail cytokinesis C_LI

cell biology↗

bioRxiv2022

Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA): a live-imaging method for detecting epithelial barrier breaches with spatiotemporal precision

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance (TER), which measures the ion flux across epithelia, or by adding traceable macromolecules to one side of the epithelium and monitoring their passage to the other side. While these methods effectively measure changes to global barrier function, they are not sensitive enough to detect local or transient disruptions in the barrier, and they do not reveal the location of barrier breaches within the context of cell or tissue morphology. Therefore, we developed a method that we named Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction (TJ) leaks with high spatial and temporal resolution (Stephenson et al., 2019; Varadarajan et al., 2021). Here, we present expanded applications for ZnUMBA. First, we show that ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin dynamics following laser injury of the junction. We also demonstrate that ZnUMBA can be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby Canine Kidney II (MDCK II) epithelial cells. ZnUMBA is a powerful and flexible method that, with some optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

cell biology↗

bioRxiv2022

p115RhoGEF activates RhoA to support tight junction maintenance and remodeling

In vertebrates, epithelial cell-cell junctions must rapidly remodel to maintain barrier function as cells undergo dynamic shape-change events. Consequently, localized leaks sometimes arise within the tight junction (TJ) barrier, which are repaired by short-lived activations of RhoA, called "Rho flares". However, what activates RhoA at leak sites remains unknown. Here, we asked which guanine nucleotide exchange factor (GEF) localizes to TJs to initiate Rho activity at Rho flares. We find that p115RhoGEF locally activates Rho flares at sites of barrier leaks and TJ loss. Knockdown of p115RhoGEF leads to diminished Rho flare intensity and impaired TJ remodeling. p115RhoGEF knockdown also decreases junctional active RhoA levels, thus compromising the apical actomyosin array and junctional complex. Furthermore, p115RhoGEF is necessary to preserve global TJ barrier function by promoting local leak repair and preventing repeating leaks. In all, our work demonstrates a central role for p115RhoGEF in activating junctional RhoA to preserve barrier function and direct local TJ remodeling.

cell biology↗

bioRxiv2022

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD, to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in the development of a broad class of novel therapies to slow cone degeneration.

neuroscience↗

bioRxiv2021

Mechanosensitive calcium signaling promotes epithelial tight junction remodeling by activating RhoA

Epithelia maintain an effective barrier by actively remodeling cell-cell junctions in response to mechanical stimuli. Cells often respond to mechanical stress through activation of RhoA and dynamic remodeling of actomyosin. Previously, we found that local leaks in the epithelial tight junction barrier are rapidly repaired by localized, transient activation of RhoA, a process we termed "Rho flares", but how Rho flares are initiated remains unknown. Here, we discovered that intracellular calcium flashes occur in Xenopus laevis embryonic epithelial cells undergoing rapid remodeling of tight junctions via activation of Rho flares. Calcium flashes originate at the site of leaks and propagate into the cell. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSC) reduced the amplitude of calcium flashes and diminished the activation of Rho flares. Furthermore, MSC-dependent calcium influx was necessary to maintain global barrier function by regulating repair of local tight junction proteins through efficient contraction of junctions. Collectively, we propose that MSC-dependent calcium flashes are an important mechanism allowing epithelial cells to sense and respond to local leaks induced by mechanical stimuli.

cell biology↗

bioRxiv2021

Rho and F-actin self-organize within an artificial cell cortex

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division1,2. During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability"3-7. In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho8,9. These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis, while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation10. In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics11. This reconstituted system spontaneously develops two distinct dynamic patterns: singular excitable Rho and F-actin waves and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the cortical excitability previously characterized in vivo9. These findings directly support the longstanding speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics. HighlightsO_LIAn artificial cell cortex comprising Xenopus egg extract on a supported lipid bilayer self-organizes into complex, dynamic patterns of active Rho and F-actin C_LIO_LIWe identified two types of reconstituted cortical dynamics - excitable waves and coherent oscillations C_LIO_LIReconstituted waves and oscillations require Rho activity and F-actin polymerization C_LI

cell biology↗

bioRxiv2021

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called Tiled-ClickSeq, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

molecular biology↗

bioRxiv2019

Transcriptional analysis of sodium valproate in a serotonergic cell line reveals gene regulation through both HDAC inhibition-dependent and independent mechanisms

Sodium valproate (VPA) is a histone deacetylase (HDAC) inhibitor, widely prescribed in the treatment of bipolar disorder, and yet the precise modes of therapeutic action for this drug are not fully understood. After exposure of the rat serotonergic cell line RN46A to VPA, RNA-sequencing (RNA-Seq) analysis showed widespread changes in gene expression. Analysis by multiple pipelines revealed as many as 230 genes were significantly upregulated and 72 genes were significantly downregulated. A subset of 23 differentially expressed genes was selected for validation using the nCounter(R) platform, and of these we obtained robust validation for ADAM23, LSP1, MAOB, MMP13, PAK3, SERPINB2, SNAP91, WNT6, and ZCCHC12. We investigated the effect of lithium on this subset and found four genes, CDKN1C, LSP1, SERPINB2 and WNT6 co-regulated by lithium and VPA. We also explored the effects of other HDAC inhibitors and the VPA analogue valpromide on the subset of 23 selected genes. Expression of eight of these genes, CDKN1C, MAOB, MMP13, NGFR, SHANK3, VGF, WNT6 and ZCCHC12, was modified by HDAC inhibition, whereas others did not appear to respond to several HDAC inhibitors tested. These results suggest VPA may regulate genes through both HDAC-dependent and independent mechanisms. Understanding the broader gene regulatory effects of VPA in this serotonergic cell model should provide insights into how this drug works and whether other HDACi compounds may have similar gene regulatory effects, as well as highlighting molecular processes that may underlie regulation of mood.

genomics↗

bioRxiv2019

Promoting neuronal outgrowth using ridged scaffolds coated with extracellular matrix proteins.

Spinal cord injury (SCI) results in cell death, demyelination, and axonal loss. The spinal cord has a limited ability to regenerate and current clinical therapies for SCI are not effective in helping promote neurologic recovery. We have developed a novel scaffold biomaterial that is fabricated from the biodegradable hydrogel oligo[poly(ethylene glycol)fumarate] (OPF). We have previously shown that positively charged OPF scaffolds (OPF+) in an open spaced, multichannel design can be loaded with Schwann cells to support axonal generation and functional recovery following SCI. We have now developed a hybrid OPF+ biomaterial that increases the surface area available for cell attachment and that contains an aligned microarchitecture and extracellular matrix (ECM) proteins to better support axonal regeneration. OPF+ was fabricated as 0.08 mm thick sheets containing 100 m high polymer ridges that self-assembles into a spiral shape when hydrated. Laminin, fibronectin, or collagen I coating promoted neuron attachment and axonal outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and neurites aligned in the direction of the ridge. Schwann cells seeded on laminin coated OPF+ sheets aligned along the ridges over a 6-day period and could myelinate dorsal root ganglion neurons over 4 weeks. The OPF+ sheets support axonal regeneration when implanted into the transected spinal cord. This novel scaffold design, with closer spaced ridges and Schwann cells is a novel biomaterial construct to promote regeneration after SCI.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC=\"FIGDIR/small/788539v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1ea0ae3org.highwire.dtl.DTLVardef@70e1f7org.highwire.dtl.DTLVardef@1034b77org.highwire.dtl.DTLVardef@14f784d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Nanopore sequencing of the pharmacogene CYP2D6 allows simultaneous haplotyping and detection of duplications

BackgroundThe accurate genotyping of CYP2D6 is hindered by the very polymorphic nature of the gene, high homology with its pseudogene CYP2D7, and the occurrence of structural variations. Long read sequencing offers the promise of overcoming some of these challenges, along with the advantage of straightforward variant phasing. We have established methods for sequencing and analysis of DNA amplicons containing the whole CYP2D6 gene, using the GridION nanopore sequencer.\n\nMaterials and methodsSeven reference and 25 clinical samples covering various haplotypes including gene duplication were barcoded and sequenced over two sequencing runs. Sequenced raw reads were analyzed using a pipeline of bioinformatics tools including two mapping tools and two variant calling tools.\n\nResultsUsing minimap2 and nanopolish (mapping and variant calling tools respectively) resulted in the most accurate variant detection. Haplotypes of 52 alleles could be matched accurately to known alleles or subvariants, while the remaining 12 alleles being assigned as novel star (*) allele of novel subvariants of known alleles in the PharmVar CYP2D6 haplotype database. Allele duplication could be detected by analyzing the allelic balance between the sample haplotypes.\n\nConclusionNanopore sequencing of CYP2D6 offers a high throughput method for genotyping, accurate haplotyping, and detection of new variants and duplicated alleles.

genetics↗

bioRxiv2019

A Multiplex Pharmacogenetics Assay using the MinION Nanopore Sequencing Device

AimThe MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. We developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, two commonly used drugs that show response variability due to genetic polymorphisms. Materials & MethodsSix reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting key variants. These products were then barcoded to enable sample multiplexing. Three variant calling tools were used to compare genotyping accuracy. Results and ConclusionsAll but three samples were successfully sequenced and genotyped. Nanopolish software achieved accuracy > 90 % for all except one variant. While minor mis-genotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays are possible on the MinION sequencing device.

genetics↗