Biology subjects

Johnson, B. K.

Publications and source records attributed to Johnson, B. K..

17 recordsLinked to original sources

bioRxiv2026

Leveraging Human Pangenome for Improved Somatic Variant Detection

Somatic variant detection is technically challenging due to low variant allele fractions, the confounding presence of germline variation, and reference bias. Linear references such as GRCh38 miss sample-specific variation, causing misalignments and incorrect variant calls. Although telomere-to-telomere donor-specific assemblies (DSAs) accurately represent individual genomes, their application is limited by cost and technical barriers. Alternatively, the graph-based human pangenome provides a scalable framework to improve read alignment and perform genome inference. Here, we benchmarked somatic variant detection using GRCh38, graph-based pangenomes, and pangenome-inferred DSAs with a HapMap mixture dataset and the COLO829 melanoma cell line. Pangenome-guided alignment improves read mapping and somatic variant calling accuracy. Furthermore, personalized pangenomes partially reconstruct donor-specific genomic content, improving accuracy, reducing germline contamination, and enabling detection of events in loci absent or poorly represented in GRCh38. These findings demonstrate that graph-based and personalized pangenomes are effective strategies for enhancing somatic variant detection compared with GRCh38.

genomics↗

bioRxiv2025

A Pangenomic Method for Establishing a Somatic Variant Detection Resource in HapMap Mixtures

Somatic mosaicism is essential in human biology and disease, yet robust benchmarks are scarce. The SMaHT Consortium mixed six HapMap cell lines to create artificial somatic variants spanning 0.25% to 16.5% variant allele fractions. We developed a technology-agnostic method that builds pangenome graphs from individual assemblies to create unified benchmarking sets: > 6M single-nucleotide variants, 1.8M small insertions/deletions, 49K structural variations, and 10K mobile element insertions across autosomes, X, and mitochondrial chromosomes. We validated the variants using ultra-deep simulated reads and developed a binomial-based model to estimate coverage requirements for variant detection. Evaluating multiple callers showed CHM13 alignment improves structural variant detection and offers advantages in difficult-to-map regions compared to GRCh38. Systematic characterization showed regions with low detection rate are enriched in centromeres, satellite sequences, tandem repeats, and falsely duplicated genes. This accurate, versatile resource enables systematic evaluation of somatic variant detection technologies.

genomics↗

bioRxiv2025

Tranquillyzer: A Flexible Neural Network Framework for Structural Annotation and Demultiplexing of Long-Read Transcriptomes

Long-read single-cell RNA sequencing using platforms such as Oxford Nanopore Technologies (ONT) enables full-length transcriptome profiling at single-cell resolution. However, high sequencing error rates, diverse library architectures, and increasing dataset scale introduce major challenges for accurately identifying cell barcodes (CBCs) and unique molecular identifiers (UMIs) - key prerequisites for reliable demultiplexing and deduplication, respectively. Existing pipelines rely on hard-coded heuristics or local transition rules that cannot fully capture this broader structural context and often fail to robustly interpret reads with indel-induced shifts, truncated segments, or non-canonical element ordering. We introduce Tranquillyzer (TRANscript QUantification In Long reads-anaLYZER), a flexible, architecture-aware deep learning framework for processing long-read single-cell RNA-seq data. Tranquillyzer employs a hybrid neural network architecture and a global, context-aware design, and enables precise identification of structural elements - even when elements are shifted, partially degraded, or repeated due to sequencing noise or library construction variability. In addition to supporting established single-cell protocols, Tranquillyzer accommodates custom library formats through rapid, one-time model training on user-defined label schemas, typically completed within a few hours on standard GPUs. Additional features such as scalability across large datasets and comprehensive visualization capabilities further position Tranquillyzer as a flexible and scalable framework solution for processing long-read single-cell transcriptomic datasets.

bioinformatics↗

bioRxiv2025

Synthbar: A Lightweight Tool for Adding Synthetic Barcodes to Sequencing Reads

Preparation of single-cell sequencing libraries includes adding nucleotide barcodes to assist with pooling samples or cells together for sequencing. The popularity of droplet-based single-cell protocols has spurred the development of computational tools that expect the read structure of the assay to include a cell barcode (CB). Microwell plate-based protocols, such as the Switching Mechanism At the 5 end of the RNA Transcript (SMART) single-cell RNA sequencing (scRNA-seq) family of methods, typically do not add a CB as part of the library preparation method as there is typically one cell per well and standard unique dual indices are sufficient for multiplexing. While several tools exist to manipulate and parse varying single-cell read structures, no tool is currently available to easily add synthetic CBs to enable use of computational tooling that expects the presence of a CB, such as STARsolo, zUMIs, and Alevin. Synthbar fills this gap as a lightweight tool that is assay agnostic, can add user-defined CBs, and modify read structures. Availability and ImplementationSource code and binaries are freely available at https://github.com/jamorrison/synthbar under the MIT license. Synthbar is implemented in C and is supported on macOS and Linux. Supplementary informationSupplementary data are available at the end of the document.

bioinformatics↗

bioRxiv2024

Dietary Restriction Enhances CD8+ T Cell Ketolysis to Limit Exhaustion and Boost Anti-Tumor Immunity

Reducing calorie intake without malnutrition limits tumor progression but the underlying mechanisms are poorly understood. Here we show that dietary restriction (DR) suppresses tumor growth by enhancing CD8+ T cell-mediated anti-tumor immunity. DR reshapes CD8+ T cell differentiation within the tumor microenvironment (TME), promoting the development of effector T cell subsets while limiting the accumulation of exhausted T (Tex) cells, and synergizes with anti-PD1 immunotherapy to restrict tumor growth. Mechanistically, DR enhances CD8+ T cell metabolic fitness through increased ketone body oxidation (ketolysis), which boosts mitochondrial membrane potential and fuels tricarboxylic acid (TCA) cycle-dependent pathways essential for T cell function. T cells deficient for ketolysis exhibit reduced mitochondrial function, increased exhaustion, and fail to control tumor growth under DR conditions. Our findings reveal a critical role for the immune system in mediating the anti-tumor effects of DR, highlighting nutritional modulation of CD8+ T cell fate in the TME as a critical determinant of anti-tumor immunity.

immunology↗

bioRxiv2024

Bone marrow microenvironment signatures associate with patient survival after guadecitabine and atezolizumab therapy in HMA-resistant MDS

Almost 50% of patients with myelodysplastic syndrome (MDS) are refractory to first-line hypomethylating agents (HMAs), which presents a significant clinical challenge considering the lack of options for salvage. Past work revealed that immune checkpoint molecules on peripheral myeloblasts and immune cells are up-regulated after HMA treatment. Therefore, we conducted a Phase I/II clinical trial combining guadecitabine (an HMA) and atezolizumab (an immune checkpoint inhibitor) to treat HMA-relapsed or refractory (HMA-R/R) MDS patients. This combination therapy showed median overall survival of 15.1 months relative to historical controls (4-6 months). Here, we profiled the cell composition and gene expression signatures of cells from bone marrow aspirates from trial participants with short-term (<15 months) or long-term (>15 months) survival at single-cell resolution. Long-term survivors showed a significant reduction of immunosuppressive monocytes, and an expansion of effector lymphocytes after combination therapy. Further immune profiling suggests that gamma delta T cell activation through primed dendritic cells was associated with global interferon activation in the bone marrow microenvironment of long-term survivors. Short-term survivors exhibited elevated inflammation and senescence-like gene signatures that were not resolved by combination therapy. We propose that distinct bone marrow microenvironment features, such as senescence-associated inflammation or immunosuppressive monocyte presence, could improve patient stratification for HMA and immunotherapy combinations in HMA-R/R MDS patients.

cancer biology↗

bioRxiv2024

High-coverage allele-resolved single-cell DNA methylation profiling by scDEEP-mC reveals cell lineage, X-inactivation state, and replication dynamics

DNA methylation is a relatively stable epigenetic mark with important roles in development and disease.1 Since cell-to-cell variation in epigenetic programming can reflect important differences in cell state and fate, it is clear that single-cell methods are essential to understanding this key epigenetic mark in heterogeneous tissues. Existing single-cell whole-genome bisulfite sequencing (scWGBS) methods2 have significant shortcomings, including very low CpG coverage3-7 or inefficient library generation requiring extremely deep sequencing.8 These methods offer limited insight into focal regulatory regions and generally preclude direct cell-to-cell comparisons. To address these shortcomings, we have developed an improved method, scDEEP-mC (single-cell Deep and Efficient Epigenomic Profiling of methyl-C). We show that high-coverage promoter methylation profiling by scDEEP-mC can identify multiple cell types, while allele-resolved methylation calls allow assessment of X-inactivation state in single cells and identification of transcription factor binding sites (TFBS) enriched for hemi-methylation. We also use scDEEP-mC to profile single-cell copy number alterations, identify actively replicating cells, and track DNA methylation dynamics during and after DNA replication. The high coverage of scDEEP-mC creates an exceptional opportunity to explore DNA methylation biology in individual cells.

genomics↗

bioRxiv2023

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of SignificanceWe developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

cancer biology↗

bioRxiv2022

Carbon dioxide regulates Mycobacterium tuberculosis PhoPR signaling and virulence

The Mycobacterium tuberculosis (Mtb) two-component regulatory system, PhoPR, is implicated in pH-sensing within the macrophage because it is strongly induced by acidic pH both in vitro and the macrophage phagosome. The carbonic anhydrase (CA) inhibitor ethoxzolamide (ETZ) inhibits PhoPR signaling supporting the hypothesis that CO2 may also play a role in regulating PhoPR. Here, we show that increasing CO2 concentration induces PhoPR signaling, and its induction is independent of medium pH. We also show that at acidic pH 5.7, a normally strong inducer of PhoPR signaling, that increasing CO2 from 0.5% to 5% further induces the pathway. Based on these findings, we propose that PhoPR functions as a CO2 sensor. Mtb has three CA (CanA, CanB, and CanC) and using CRISPR interference knockdowns and gene deletion mutants, we assessed which CAs regulate PhoPR signaling and virulence. We first examined if CA played a role in Mtb pathogenesis and observed that CanB was required for virulence in macrophages, where the knockdown strain had ~1 log reduction in virulence. To further define the interplay of CO2 and Mtb signaling, we conducted transcriptional profiling experiments at varying pH and CO2 concentrations. As hypothesized, we observed the induction of PhoPR at acidic pH is dependent on CO2 concentration, with a subset of core PhoPR regulon genes dependent on both 5% CO2 and acidic pH for their induction, including expression of the ESX-1 secretion system. Transcriptional profiling also revealed core CO2 responsive genes that were differentially expressed independently of the PhoPR regulon or the acidic pH-inducible regulon. Notably, genes regulated by a second two component regulatory system, TrcRS, are associated with adaptation to changes in CO2.

microbiology↗

bioRxiv2022

STORM-seq Reveals Differentiation Trajectories of Primary Human Fallopian Tube Epithelium

Despite significant advances, current single-cell RNA sequencing (scRNA-seq) technologies often struggle with accurately detecting non-coding transcripts, achieving full-length RNA coverage, and/or resolving transcript-level complexity. Many are also difficult to implement or inaccessible without specialized liquid handlers, further limiting their utility. We present Single-cell TOtal RNA-seq Miniaturized (STORM-seq), a random- hexamer primed, ribo-reduced single-cell total RNA sequencing (sc-total-RNA-seq) protocol using standard laboratory equipment. Adapted as a kit, STORM-seq constructs sequence-ready libraries in one working day, producing the highest complexity scRNA- seq libraries to-date, robustly measuring transcript isoforms and clinically relevant gene fusions in single cells. STORM-seq faithfully reconstructs expression profiles of locus- level transposable elements (TEs), and provides high-resolution profiling of transient, low- abundance enhancer RNAs (eRNAs), offering a powerful tool to dissect single-cell gene regulatory networks in unprecedented detail. Applied to human fallopian tube epithelium, the improved transcriptional resolution reveals a putative progenitor-like population and intermediate cell states, shaped by TEs and non-coding RNAs.

molecular biology↗

bioRxiv2021

Compartmap enables inference of higher-order chromatin structure in individual cells from scRNA-seq and scATAC-seq

Single-cell profiling of chromatin structure remains a challenge due to cost, throughput, and resolution. We introduce compartmap to reconstruct higher-order chromatin domains in individual cells from transcriptomic (RNAseq) and epigenomic (ATACseq) assays. In cell lines and primary human samples, compartmap infers higher-order chromatin structure comparable to specialized chromatin capture methods, and identifies clinically relevant structural alterations in single cells. This provides a common lens to integrate transcriptional and epigenomic results, linking higher-order chromatin architecture to gene regulation and to clinically relevant phenotypes in individual cells.

genomics↗

bioRxiv2021

Games of risk and reward in carnivore communities

Mesopredator release theory suggests that dominant predators suppress subordinate carnivores and ultimately shape community dynamics, but the assumption that subordinate species are only negatively affected ignores the possibility of facilitation through scavenging. We examined the interplay within a carnivore community consisting of cougars, coyotes, black bears, and bobcats using contemporaneous Global Positioning System telemetry data from 51 individuals, diet analysis from 972 DNA-metabarcoded scats, and data from 128 physical investigations of cougar kill sites, 28 of which were monitored with remote cameras. Resource provisioning from competitively-dominant cougars to coyotes through scavenging was so prolific as to be an overwhelming determinant of coyote behavior, space use, and resource acquisition. This was evident via strong attraction of coyotes to cougar kill sites, frequent scavenging of cougar-killed prey, and coyote diets that nearly matched cougars in the magnitude of ungulate consumption. Yet coyotes were often killed by cougars and used space to minimize encounters, complicating the fitness benefits gained from scavenging. We estimated that 23% (95% CI: 8-55%) of the coyote population in our study area was killed by cougars annually suggesting that coyote interactions with cougars are a complex behavioral game of risk and reward. In contrast, we found no indication that bobcat space use or diet was influenced by cougars. Black bears avoided cougars, but there was no evidence of attraction to cougar kill sites, and much lower levels of ungulate consumption and carcass visitation than for coyotes. Interspecific interactions among carnivores are multifaceted encompassing both suppression and facilitation. Significance StatementAn incomplete understanding of the total influence competitively-dominant predators exert on subordinate species hinders our ability to anticipate the effects that changing carnivore populations will have in ecological communities. Here we show that cougars are the architects of a complex behavioral game of risk and reward, as subordinate or co-occurring carnivores are both provisioned and preyed on by the dominant predators. Each co-occurring carnivore species considered here employed a different strategy to approach the risk-reward tradeoff suggesting there are multiple viable solutions to the game. By not considering the multitude of effects dominant predators have on other carnivores, we are missing important linkages in terrestrial food webs.

ecology↗

bioRxiv2020

Integrating spatial capture-recapture models with variable individual identifiability

Spatial capture-recapture (SCR) models have become the preferred tool for estimating densities of carnivores. Within this family of models are variants requiring identification of all individuals in each encounter (SCR), a subset of individuals only (generalized spatial mark-resight, gSMR), or no individual identification (spatial count or spatial presence-absence). Although each technique has been shown through simulation to yield unbiased results, the consistency and relative precision of estimates across methods in real-world settings are seldom considered. We tested a suite of models ranging from those only requiring detections of unmarked individuals to others that integrate remote camera, physical capture, genetic, and global positioning system (GPS) data into a hybrid model, to estimate population densities of black bears, bobcats, cougars, and coyotes. For each species we genotyped fecal DNA collected with detection dogs during a 20-day period. A subset of individuals from each species was affixed with GPS collars bearing unique markings and resighted by remote cameras over 140 days contemporaneous with scat collection. Camera-based gSMR models produced density estimates that differed by less than 10% from genetic SCR for bears, cougars, and coyotes once important sources of variation (sex or behavioral status) were controlled for. For bobcats, SCR estimates were 33% higher than gSMR. The cause of the discrepancies in estimates was likely attributable to challenges designing a study compatible for species with disparate home range sizes and the difficulty of collecting sufficient data in a timeframe in which demographic closure could be assumed. Unmarked models estimated densities that varied greatly from SCR, but estimates became more consistent in models wherein more individuals were identifiable. Hybrid models containing all data sources exhibited the most precise estimates for all species. For studies in which only sparse data can be obtained and the strictest model assumptions are unlikely to be met, we suggest researchers use caution making inference from models lacking individual identity. For best results, we further recommend the use of methods requiring at least a subset of the population is marked and that multiple datasets are incorporated when possible.

ecology↗

bioRxiv2019

Direct therapeutic targeting of SWI/SNF induces epigenetic reprogramming and durable tumor regression in rhabdoid tumor

PurposeRhabdoid tumor is a pediatric cancer characterized by the biallelic inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 inactivation leads to SWI/SNF redistribution to favor a proliferative dedifferentiated cellular state. Although this deletion is the known oncogenic driver, SWI/SNF therapeutic targeting remains a challenge. Experimental DesignWe define a novel epigenetic mechanism for mithramycin using biochemical fractionation, chromatin immunoprecipitation sequencing (ChIP-seq), and a dual spike-in assay for transposase accessible chromatin sequencing (ATAC-seq). We correlate epigenetic reprogramming with changes with chromatin A/B compartments and promoter accessibility with chromHMM models and RNA-seq. Finally, we demonstrate durable, marked tumor response in an intramuscular rhabdoid tumor xenograft model. ResultsHere we show mithramycin and a second-generation analogue EC8042 evict mutated SWI/SNF from chromatin and are effective in rhabdoid tumor. SWI/SNF blockade triggers chromatin compartment remodeling and promoter reprogramming leading to differentiation and amplification of H3K27me3, the catalytic mark of PRC2. Treatment of rhabdoid rumor xenografts with EC8042 leads to marked, durable tumor regression and differentiation of the tumor tissue into benign mesenchymal tissue, including de novo bone formation. ConclusionOverall, this study identifies a novel therapeutic candidate for rhabdoid tumor and an approach that may be applicable to the 20% of cancers characterized by mutated SWI/SNF. STATEMENT OF TRANSLATIONAL RELEVANCEThere is a tremendous need for novel therapeutic approaches for rhabdoid tumor and the more than 20% of human cancers characterized by dysregulation of the SWI/SNF chromatin remodeling complex. While approaches to target associated complexes, such as PRC2, known to be influenced by dysregulated SWI/SNF are currently being evaluated in the clinic, the direct therapeutic targeting of SWI/SNF has not been explored. Here we identify an inhibitor of SWI/SNF and thoroughly explore the therapeutic development of this compound from a mechanistic and translational perspective thus providing insight into the targeting of this complex as well as a dose, schedule, and biomarker of target inhibition that is immediately clinically translatable.

cancer biology↗

bioRxiv2019

Distinctive epigenomic alterations in NF1-deficient cutaneous and plexiform neurofibromas drive differential MKK/P38 signaling

Benign peripheral nerve sheath tumors are the clinical hallmark of Neurofibromatosis Type 1. They account for substantial morbidity in NF1 and are difficult to manage. Cutaneous (CNF) and plexiform neurofibromas (PNF) share identical histology, but maintain different growth rates and risk of malignant conversion. The reasons for their disparate clinical behavior are not well explained on the basis of recent genome or transcriptome profiling studies. We hypothesized that CNFs and PNFs are epigenetically distinct tumor types that exhibit differential signaling due to genome-wide and site-specific methylation events. We interrogated the methylation profiles of 45 CNFs and 17 PNFs (Illumina EPIC 850K) using normal tissue controls from NF1 subjects. Based on these profiles, we confirm that CNFs and PNFs are epigenetically distinct tumors with broad differences in higher order chromatin states, and specific methylation events altering genes involved in key biological and cellular processes such as inflammatory mediator regulation of TRP channels, RAS/MAPK signaling, actin cytoskeleton rearrangement, and oxytocin signaling. Based our identification of 2 separate DMRs associated with alternative leading exons in MAP2K3, we demonstrate differential RAS/MKK3/P38 signaling between CNFs and PNFs. Epigenetic reinforcement of RAS/MKK/P38 was a defining characteristic of CNFs leading to pro-inflammatory signaling and chromatin conformational changes, whereas PNFs signaled predominantly through RAS/ERK. Tumor size also correlated with specific CpG methylation events. Taken together, these findings confirm that epigenetic regulation of RAS signaling fates accounts for observed differences in CNF and PNF clinical behavior. CNFs may also respond differently than PNFs to RAS-targeted therapeutics raising the possibility of targeting P38-mediated inflammation for CNF treatment.

genomics↗

bioRxiv2019

Elizabethkingia anophelis response to iron stress: physiologic, genomic, and transcriptomic analyses

Elizabethkingia anophelis bacteria encounter fluxes of iron in the midgut of mosquitoes, where they live as symbionts. They also establish bacteremia with severe clinical manifestations in humans, and live in water service lines in hospitals. In this study, we investigated the global gene expression responses of E. anophelis to iron fluxes in the midgut of female Anopheles stephensi mosquitoes fed sucrose or blood, and in iron-poor or iron-rich culture conditions. Of 3,686 transcripts revealed by RNAseq technology, 218 were upregulated while 112 were down-regulated under iron-poor conditions. Most of these differentially expressed genes (DEGs) were enriched in functional groups assigned within \"biological process,\" \"cell component\" and \"molecular function\" categories. E. anophelis possessed 4 iron/heme acquisition systems. Hemolysin gene expression was significantly repressed when cells were grown under iron-rich or high temperature (37{degrees}C) conditions. Furthermore, hemolysin gene expression was down-regulated after a blood meal, indicating that E. anophelis cells responded to excess iron and its associated physiological stress by limiting iron loading. By contrast, genes encoding respiratory chain proteins were up-regulated under iron-rich conditions, allowing these iron-containing proteins to chelate intracellular free iron. In vivo studies showed that growth of E. anophelis cells increased 3-fold in blood-fed mosquitoes over those in sucrose-fed ones. Deletion of aerobactin synthesis genes led to impaired cell growth in both iron-rich and iron-poor media. Mutants showed more susceptibility to H2O2 toxicity and less biofilm formation than did wild-type cells. Mosquitoes with E. anophelis experimentally colonized in their guts produced more eggs than did those treated with erythromycin or left unmanipulated, as controls. Results reveal that E. anophelis bacteria respond to varying iron concentration in the mosquito gut, harvest iron while fending off iron-associated stress, contribute to lysis of red blood cells, and positively influence mosquito host fecundity.

microbiology↗

bioRxiv2016

Acidic pH-dependent depletion of Mycobacterium tuberculosis thiol pools potentiates antibiotics and oxidizing agents

Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH and reactive oxygen species (ROS) during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the acidic pH-dependent compound AC2P36 depletes intracellular thiol pools, sensitizes Mtb to killing by acidic pH, and potentiates the bactericidal activity of isoniazid, clofazimine, and oxidizing agents. We show that the pHdependent activity of AC2P36 is associated with metabolic stress at acidic pH and a pHdependent accumulation of intracellular ROS. Mechanism of action studies show that AC2P36 directly depletes Mtb thiol pools. These data support a model where chemical depletion of Mtb thiol pools at acidic pH enhances sensitivity to oxidative damage, resulting in bacterial killing and potentiation of antibiotics.

microbiology↗