Biology subjects

Raine, A.

Publications and source records attributed to Raine, A..

13 recordsLinked to original sources

bioRxiv2026

upSPLAT: Early-Barcoded Library Preparation for Cost-Effective Population-Scale Genomics

Advances in high-throughput sequencing have substantially reduced sequencing costs, yet library preparation remains a major financial and logistical bottleneck, particularly for high-throughput applications or low-quality DNA inputs. Here, we introduce upscaled Splinted Ligation Adapter Tagging (upSPLAT), a library preparation strategy that combines early sample barcoding with single-stranded splinted ligation to enable highly multiplexed pooled sequencing at substancially reduced cost. upSPLAT supports flexible high-plex pooling and reduces per-sample library preparation costs by approximately 10-fold compared to conventional workflows. By leveraging single-strand ligation, upSPLAT is compatible with a wide range of DNA inputs, including degraded, damaged or denatured double stranded DNA, bisulfite or enzymatically converted DNA, and viral single-stranded DNA. We present two complementary workflows and evaluate their performance across multiple species and DNA qualities, demonstrating robust demultiplexing, uniform sample representation, and low barcode cross-assignment. Together, upSPLAT provides a scalable, cost-effective solution for sequencing-based studies requiring large sample numbers while preserving individual-level information.

genomics↗

bioRxiv2025

Circulating cell-free RNA reflects inflammatory and airway remodeling signatures in an equine model of asthma

Circulating cell-free RNA (cfRNA) offers a minimally invasive means to monitor dynamic gene expression across tissues, providing a promising source of biomarkers for inflammatory diseases. Equine asthma, a prevalent respiratory disorder, represents a valuable translational model for the human condition. Here, we present the first transcriptomic analysis of plasma cfRNA from asthmatic and healthy horses, alongside comparative profiling of bronchoalveolar lavage and whole blood. Tissue-of-origin deconvolution revealed asthma-associated immune cell redistribution, characterized by reduced B-cell and increased T-cell signatures that were undetectable in the conventional sampling. Differential expression analysis identified up-regulation of key alarmins and transcripts linked to airway remodeling and immune activation. Cross-compartment correlation demonstrated that cfRNA captures distinct yet biologically relevant molecular signatures consistent with asthma-related pathways. These results establish cfRNA as a sensitive complementary approach for characterizing molecular perturbations in asthma pathogenesis and highlight its potential for biomarker discovery across inflammatory and allergic airway diseases.

genomics↗

bioRxiv2024

Exploring a pico-well based scRNA-seq method (HIVE) for simplified processing of equine bronchoalveolar lavage cells

Single-cell RNA sequencing (scRNA-seq) is a valuable tool for investigating cellular heterogeneity in diseases such as equine asthma (EA). This study evaluates the HIVE scRNA-seq method, a pico-well-based technology, for processing bronchoalveolar lavage (BAL) cells from horses with EA. The HIVE method offers practical advantages, including compatibility with both field and clinical settings, as well as a gentle workflow suited for handling sensitive cells. Our results show that the major cell types in equine BAL were successfully identified; however, the proportions of T cells and macrophages deviated from cytological expectations, with macrophages being overrepresented and T cells underrepresented. Despite these limitations, the HIVE method confirmed previously identified T cell and macrophage subpopulations and defined other BAL cell subsets. However, compared to previous studies T helper subsets were less clearly defined. Additionally, consistent with previous scRNA-seq studies, the HIVE method detected fewer granulocytes and mast cells than anticipated in the total BAL samples. Nevertheless, applying the method to purified mast cells recovered an expected number of cells. A small set of eosinophils were also detected which have not been characterized in earlier studies. In summary these findings suggest that while the HIVE method shows promise for certain applications, further optimization is needed to improve the accuracy of cell type representation, particularly for granulocytes and mast cells, in BAL samples.

genomics↗

bioRxiv2024

Mapping the Spatial Proteome of Leukemia Cells Undergoing Fludarabine Treatment

Recent advancements in spatial biology have revolutionized our understanding of the organization and functional dynamics of cells and tissues. In this study, we applied Molecular Pixelation (MPX), a single-cell spatial proteomics assay, to investigate the modulation of the cell surface proteome in an in vitro drug screening model using the ETV6::RUNX1 acute lymphoblastic leukemia (ALL) cell line, Reh. Specifically, we focused on the in vitro response to fludarabine, a chemotherapeutic agent used prior to allogenic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy in high-risk, refractory, or relapsed ALL patients. Using MPX, we quantified changes in protein abundance, spatial distribution, and colocalization of 76 targeted cell surface proteins in Reh cells before and after fludarabine treatment. Our analysis revealed 25 proteins with altered abundance, 24 proteins with increased polarity, and 138 protein pairs with modified colocalization following treatment. Notably, the tetraspanins CD82 and CD53, which are known for their roles in chemotherapy resistance, exhibited increased abundance, polarization, and colocalization post-treatment, suggesting their potential as a therapeutic scaffold. These findings underscore the unique ability of spatially resolved single-cell proteomics to uncover nuanced cellular responses that would otherwise remain undetected.

cancer biology↗

bioRxiv2023

COMPARISON OF HIGH-THROUGHPUT SINGLE-CELL RNA-SEQ METHODS FOR EX VIVO DRUG SCREENING

Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27,327 cells are freely available to extend to future scRNA-seq methodological comparisons.

genomics↗

bioRxiv2023

A complete digital karyotype of the B-cell leukemia REH cell line resolved by long-read sequencing

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6-RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-year-old at relapse, the cell line offers a practical model for the study of high-risk pediatric B-ALL patients. In recent years, short-read DNA and RNA sequencing have emerged as a complement to analog karyotyping techniques in the resolution of structural variants in an oncological context. However, it is challenging to create a comprehensive digital karyotype of a genome with these techniques alone. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole genome sequencing (WGS), IsoSeq RNA-sequencing, and short-read sequencing to create a detailed digital karyotype of the REH cell line. WGS refined the breakpoints of known aberrations and clarified the molecular traits of disrupted ALL-associated genes BTG1 and TBL1XR1, as well as the glucocorticoid receptor NR3C1. Several previously underreported structural variants were also uncovered, including deletions affecting the ALL-associated genes VPREB1 and NFATC1. Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community. KEY POINTSO_LIA complete digital karyotype of the REH cell line was produced with short- and long-read DNA and RNA sequencing technologies. C_LIO_LIThe study enabled precise identification of structural variants, and the fusion genes expressed as the result of these variants. C_LI

cancer biology↗

bioRxiv2022

An equine Endothelin 3 cis-regulatory variant links blood pressure modulation to elite racing performance

A previous selective sweep analysis of horse racing performance revealed a 19.6 kb candidate region approximately 50 kb downstream of the Endothelin 3 (EDN3) gene. EDN3 and other endothelin family members are associated with blood pressure regulation in humans and other species, but similar association studies in horses are lacking. We hypothesized that the sweep region includes a regulatory element acting on EDN3 transcription, ultimately affecting blood pressure regulation and athletic performance in horses. Selective sweep fine- mapping identified a 5.5 kb haplotype of 14 SNPs shared within Coldblooded trotters (CBT) and Standardbreds (SB). Most SNPs overlapped potential transcription factor binding sites, and haplotype analysis showed significant association with all tested performance traits in CBTs and earnings in SBs. From those, two haplotypes were defined: an elite performing haplotype (EPH) and a sub-elite performing haplotype (SPH). While the majority of SNPs in the haplotype were part of the standing variation already found in pre-domestication horses, there has been an increase in the frequencies of the alternative alleles during the whole history of horse domestication. Horses homozygous for EPH had significantly higher plasma levels of EDN3, lower levels of EDN1, and lower exercise-related blood pressure compared to SPH homozygous horses. Additionally, a global proteomic analysis of plasma from EPH or SPH homozygous horses revealed higher levels of proteins involved in pathways related to immune response and complement activation in the SPH horses. This is the first study to demonstrate an association between the EDN3 gene, blood pressure regulation, and athletic performance in horses. The results advance our understanding of the molecular genetics of athletic performance, exercise-related blood pressure regulation, and biological processes activated by intense exercise. Author summaryThe horse is one of the most common species used for studying athletic performance. For centuries, horses have been used by humans for transportation, agriculture and entertainment and this has resulted in selection for various traits related to athletic performance. A previous study discovered that a genetic region close to the Endothelin3 gene was associated with harness racing performance. Endothelin3 is known to be involved in blood pressure regulation and therefore we hypothesized that this region influences blood pressure and racing performance in horses. In this study we have used additional horses and fine-mapped the candidate region and we also measured blood pressure in Coldblooded trotters during exercise. Horses with two copies of the elite-performing haplotype had higher levels of Endothelin3 in plasma, lower blood pressure and better racing performance results, compared to horses with two copies of the sub-elite performing haplotype. We also discovered that horses with the sub-elite performing haplotype had higher levels of proteins related to the immune system in plasma. This study is the first to link Endothelin3 to blood pressure regulation and performance in horses. It broadens the understanding of the biological mechanisms behind blood pressure regulation as well as inflammation and coagulation system in relation to racing performance.

genetics↗

bioRxiv2022

Single cell transcriptomics delineates the immune-cell landscape in equine lower airways and reveals upregulation of the FKBP5 gene in horses with asthma

BackgroundEquine asthma (EA) is a heterogenous, complex disease with a significant negative impact on horse welfare and performance. EA and human asthma have fundamental similarities, making EA a useful large animal disease model. Bronchoalveolar lavage (BAL) fluid provides a snapshot sample of the immune cells occupying the alveolar space and is one of the most relevant sample types for studies of chronic inflammation in the lung. In this study, we sequenced single equine BAL-cells in order to study the immune cell landscape of the respiratory tract of horses diagnosed with mild-to-moderate EA and healthy controls. ResultsScRNA-seq analysis of ~63,000 cells from eleven horses diagnosed with mild-moderate asthma (mEA) and eight healthy controls were performed using the Drop-Seq technology. We identified five major immune cell populations in equine BAL; alveolar macrophages (AM), T cells, neutrophils, mast cells and dendritic cells, as well as subtypes thereof. The cellular subtypes demonstrated herein have previously not been characterized in horses. Differential gene expression analysis revealed upregulation of genes in mEA horses, including FKBP5 and CCL24, which have previously been associated with asthma in other species. The most significantly upregulated gene across the cell types in EA was FKBP5, a chaperone protein involved in regulating the assembly, activity, and sensitivity of the glucocorticoid receptor ConclusionHerein we demonstrate the first comprehensive scRNA-seq map of the immune-cell populations in BAL from horses with asthma and healthy individuals. The glucocorticoid receptor associated protein FKBP5 was identified as a potential biomarker for EA.

genomics↗

bioRxiv2021

Environmentally-induced DNA methylation is inherited across generations in an aquatic keystone species (Daphnia magna)

Environmental stress can result in epigenetic modifications that are passed down several generations. Such epigenetic inheritance can have significant impact on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. Here, we used whole-genome bisulfite sequencing on individual water fleas (Daphnia magna) to assess whether environmentally-induced DNA methylation can persist for up to four generations. Genetically identical females were exposed to a control treatment, one of three natural stressors (high temperature, zinc, microcystin), or the methylation-inhibitor 5-azacytidine. After exposure, lines were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) between controls and F1 individuals whose mothers (and therefore they themselves as germ cells) were exposed to one of the three natural stressors. Between 46% and 58% of these environmentally-induced DMPs persisted until generation F4 without attenuation in their magnitude of differential methylation. DMPs were enriched in exons and largely stressor-specific, suggesting a possible role in environment-dependent gene regulation. In contrast, treatment with the compound 5-azacytidine demonstrated that pervasive hypo-methylation upon exposure is reset almost completely after a single generation. These results suggest that environmentally-induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of fresh-water communities. Author summaryWater fleas are important keystone species mediating eco-evolutionary dynamics in lakes and ponds. It is currently an open question in how far epigenetic inheritance contributes to the ability of Daphnia populations to adapt to environmental stress. Using a range of naturally occurring stressors and a multi-generational design, we show that environmentally-induced DNA methylation variants are stably inherited for at least four generations in Daphnia magna. The induced variation in DNA methylation are stressor-specific and almost exclusively found in exons, bearing the signatures of functional adaptations. Our findings imply that ecological adaptations of Daphnia to seasonal fluctuations can be underpinned by epigenetic inheritance of DNA methylation without changes in gene frequencies.

evolutionary biology↗

bioRxiv2021

scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods. MotivationscWGBS library preparation in a one-cell-per-library format presents practical and economical constraints to the number of cells that can be analyzed in a research project. In addition, most of the current scWGBS methods suffer from low read alignment rates. We present a scWGBS protocol which mitigates these issues, empowering single-cell DNA methylation analysis at an increased scale.

genomics↗

bioRxiv2020

The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDAs Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methylcytosine modifications. Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), one enzymatic deamination method (EMseq), targeted methylation sequencing (Illumina Methyl Capture EPIC), and single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, BWAMeth, and GemBS), we found overall high concordance between assays (R=0.87-R0.93), differences in efficency of read mapping and CpG capture and coverage, and platform performance. The data provided herein can guide continued used of these reference materials in epigenomics assays, as well as provide best practices for epigenomics research and experimental design in future studies.

genomics↗

bioRxiv2018

Refined detection and phasing of structural aberrations in pediatric acute lymphoblastic leukemia by linked-read whole genome sequencing

Structural chromosomal rearrangements that may lead to in-frame gene-fusions represent a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). However, short-read whole genome sequencing (WGS) technologies struggle to accurately identify and phase such large-scale chromosomal aberrations in cancer genomes. We therefore evaluated linked-read WGS for detection of chromosomal rearrangements in an ALL cell line (REH) and primary samples of varying DNA quality from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations (CNAs) and structural variants (SVs). Biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified additional structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. Thus, linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping or short-read WGS.

genomics↗

bioRxiv2017

Data quality of Whole Genome Bisulfite Sequencing on Illumina platforms

The powerful HiSeq X sequencers with their patterned flowcell technology and fast turnaround times are instrumental for many large-scale genomic and epigenomic studies. However, assessment of DNA methylation by sodium bisulfite treatment results in sequencing libraries of low diversity, which may impact data quality and yield. In this report we assess the quality of WGBS data generated on the HiSeq X system in comparison with data generated on the HiSeq 2500 system and the newly released NovaSeq system. We report a systematic issue with low basecall quality scores assigned to guanines in the second read of WGBS when using certain Real Time Analysis (RTA) software versions on the HiSeq X sequencer, reminiscent of an issue that was previously reported with certain HiSeq 2500 software versions. However, with the HD.3.4.0/RTA 2.7.7 software upgrade for the HiSeq X system, we observed an overall improved quality and yield of the WGBS data generated, which in turn empowers cost-effective and high quality DNA methylation studies.

genomics↗