Biology subjects

Campbell, J. D.

Publications and source records attributed to Campbell, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Trisomy 21 Perturbs Oligodendrocyte Developmental States and Oligo-Neuron Communication in Human Forebrain Assembloids

Intellectual disability is a hallmark of Down syndrome (DS), caused by the triplication of human chromosome 21. Abnormal neuronal connectivity and impaired myelination are consistently observed in DS, yet the underlying mechanisms remain poorly understood. Here, we used DS-derived isogenic induced pluripotent stem cell-derived forebrain assembloids (FAs) to investigate oligodendrocyte lineage cell (OLLC) development and neuron-glia interactions. Trisomic FAs exhibited an expansion of BCAS1+ pre-myelinating OLLCs, without a corresponding increase in myelin basic protein (MBP) expression. Transcriptomic analyses identified a euploid-enriched, trophic and maturation-supportive state (OLLC_2-TMS) and a trisomy-enriched synaptic interactions and differentiation state (OLLC_3-SID), lacking coordinated enrichment in myelination-promoting programs. Corresponding transcriptional signatures were identified in OLLCs from early postnatal human cortex. Increased excitatory neuronal contacts with pre-myelinating OLLCs accompanied ultrastructural myelin abnormalities in trisomic assembloids. Our findings suggest that trisomy redirects OLLC development toward a pre-myelinating state, uncoupling oligodendrocyte differentiation from productive myelination.

neuroscience↗

bioRxiv2026

Benchmarking of local ancestry inference with different assays and parameters

Local ancestry inference (LAI) enables high-resolution characterization of chromosomal segments inherited from distinct ancestral populations, offering unique insights into genetic architecture in admixed cohorts. While LAI is commonly performed with high-coverage whole-genome sequencing (WGS), the ability of other genotyping assays or varying sequencing depths has not been thoroughly benchmarked. In this study, we systematically evaluated the accuracy of LAI across SNP microarrays, whole-exome sequencing (WES), and ultra low-pass WGS (ULP-WGS) using diverse validation samples and state-of-the-art imputation pipelines. We show that ULP-WGS, when paired with GLIMPSE2, achieves robust accuracy at 0.25x coverage with a minimum genome window size of 0.5 centimorgans, with mean accuracy minus one standard deviation exceeding 95%. For WES, using "on-target" reads alone yields suboptimal performance, particularly for European and South Asian ancestries with accuracy less than 79.1% and 70.6%, respectively. However, incorporating "off-target" reads in WES and utilizing GLIMPSE2 substantially improved accuracy [≥]95% with a minimum window size of 0.2 centimorgans. We further evaluated formalin-fixed, paraffin-embedded (FFPE) samples and found that LAI could be performed successfully using WES data with accuracies of [≥]95% at a minimum window size of 0.5 centimorgans. In contrast, SNP microarrays did not achieve substantial accuracies at any window size ([≤]95%). Together, these results demonstrate that LAI is achievable without conventional high-coverage WGS and establish optimal parameters for LAI across platforms.

genomics↗

bioRxiv2026

A single-cell and spatial atlas of prostate cancer reveals the combinatorial nature of gene modules underlying lineage plasticity and metastasis

Prostate cancer encompasses a spectrum of disease states driven by complex cellular heterogeneity. To delineate the transcriptional programs underlying lineage plasticity and metastasis, we constructed a comprehensive single-cell atlas of 128 patients, spanning localized, castration-resistant, and metastatic disease. Lineage plasticity was prevalent in localized disease, with subsets of tumor cells adopting distinct basal-like and club-like states. Luminal-like cancer cells also displayed extensive lineage infidelity, defined not by a binary loss of identity but by the combinatorial erosion of luminal gene modules associated with higher grade and stage. In the metastatic setting, gene program association analysis (GPAS) identified a broad induction of cell-cycle gene modules across organ sites as well as an induction of organ-specific gene modules, including osteomimetic signaling in bone, neuro-migratory genes in brain, and erythroid-like transitions in liver. Neuroendocrine prostate cancers (NEPCs) were not monolithic but defined by combinations of NE-associated gene modules including a novel HES6 program. Notably, these modules were detected at intermediate levels in localized samples, suggesting molecular plasticity precedes histological transformation. We also developed a refined NE signature that could distinguish NEPC tumors more accurately than previously published signatures. Within the tumor microenvironment (TME), we observed an elevation of pro-inflammatory Th17 T-cells in African American patients and identified a rare Schwann cell population. Finally, we present PCformer, a transformer-based foundation model trained on >500,000 cells to automate cell-state classification. Together, this comprehensive atlas demonstrates the complex nature of gene modules underlying lineage infidelity and plasticity in cancer cells and highlights distinct immune and stromal populations within the tumor ecosystem.

genomics↗

bioRxiv2026

Defining mutational signatures of lung cancer-associated carcinogens through in vitro exposure of human airway epithelial cells

While distinct environmental exposures imprint unique mutational signatures on cancer genomes, the specific causal patterns for many known carcinogens remain uncharacterized in relevant human tissues. To address this gap, we developed a novel, physiologically relevant system that uses a combination of airway epithelial cells and whole genome sequencing to characterize mutational patterns induced by genotoxic carcinogens associated with lung cancer. After validating the platforms accuracy by successfully recapturing the known signature for Benzo(a)pyrene (BaP), we used this system to gain detailed insights into the types of mutations that occur with exposure to N-nitrosotris-(2-chloroethyl) urea (NTCU) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), genotoxic compounds that induce lung squamous cell carcinoma and lung adenocarcinoma in mouse models, respectively. Cells exposed to NTCU had significantly more somatic SNVs compared to control samples. An average of 82.3% of mutations in NTCU samples were attributed to a novel mutational signature distinct from those in the COSMIC database but highly correlated with recent in vivo mouse models. In contrast, NNK exposure did not demonstrate a distinct mutational pattern above background at both high and low concentrations. Ultimately, this in vitro system provides a robust platform to define causal links between environmental exposures and mutational patterns in lung cancer mutagenesis. Statement of SignificanceIn vitro exposure of N-nitrosotris-(2-chloroethyl) urea to airway epithelial cells revealed a distinct mutational signature.

bioinformatics↗

bioRxiv2025

A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins

The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4+ and CD8+ T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.

microbiology↗

bioRxiv2025

Transcriptional and functional profiles of muscarinic receptor-expressing neurons in primate lateral prefrontal and anterior cingulate cortices

Acetylcholine modulates anterior cingulate (ACC) and lateral prefrontal (LPFC) cortices for cognitive-motivational integration, via specific m1-m4 muscarinic receptors (mAChR) encoded by CHRM1-4 genes. Single-nucleus RNA sequencing and mRNA-protein histology in macaques revealed CHRM3 to be the most enriched mAChR gene in neurons, while m1 predominates at the protein level, likely due to nuclear retention of CHRM3 and cytoplasmic trafficking of CHRM1. CHRM3 and CHRM1 showed strong co-expression and functional overlap, and were transcriptomically-distinct from CHRM2, which was uniquely enriched in deep layer excitatory and PVALB+ inhibitory neurons. Although CHRM+ cell distributions were similar between areas, CHRM1-3+ excitatory neurons in ACC exhibited upregulation of synaptic plasticity genes relative to LPFC. Functional in vitro experiments confirm a more robust cholinergic-mediated decrease in excitatory:inhibitory synaptic ratio in ACC than in LPFC neurons, accompanied by compensatory changes in spine morphology. These findings highlight region-specific acetylcholine signaling essential for flexible processing, learning and memory.

neuroscience↗

bioRxiv2025

Sub-phenotypes of pneumonia defined by pulmonary histopathological features

Establishing sub-phenotypes of pneumonia based on distinct host processes will be a step towards using host-directed therapies (to complement microbe-directed therapies) more rationally and precisely. Although pneumonia is a pulmonary pathophysiology, histological changes within the lungs have not been leveraged for sub-phenotyping. We addressed this by scoring 18 histopathology features (e.g., type 2 cell hyperplasia or necrosis) across rapid autopsy lung samples from 276 elderly subjects with pneumonia. Machine learning algorithms segregated subjects into seven different sub-phenotypes of pneumonia with distinct histopathology signatures. Quantitative immunofluorescence demonstrated associations of macrophages, neutrophils, T cells, and B cells with select histology features and pulmonary pathology sub-phenotypes. Mouse models revealed corollary sub-phenotypes, although some histology features observed in human lungs were never observed in mice. By illuminating this spectrum of histopathologies and discriminating discrete sub-phenotypes of pneumonia, a foundational framework emerges for developing and using host-directed therapies for subsets of pneumonia patients.

immunology↗

bioRxiv2025

An atlas of gene programs in non-small cell lung cancer delineates novel patterns of lineage vacillation, plasticity, and aggressiveness

Combinations of molecular programs can be altered in cancer cells and contribute to tumor initiation and aggressiveness. We developed a novel framework called a Gene Program Association Study (GPAS) to create an atlas of gene co-expression modules in cancer cells from 127 primary non-small-cell-lung cancers (NSCLCs) and determine their association with clinical and histopathological features. Lung adenocarcinoma (LUAD) displayed "lineage vacillation" defined by extensive heterogeneity of modules from different alveolar cell types. Modules associated with late-stage LUAD were found in cells from early-stage tumors suggesting that aggressive phenotypes can be observed before clinical progression. Basal- and squamous-like intermediate cells were observed in the transition to invasive mucinous LUAD. In lung squamous cell carcinoma, a novel subtype was identified with lower levels of canonical squamous modules and higher levels of fibrinogen. Overall, this atlas elucidated the combinations of molecular programs in cancer cells that contribute to heterogeneity and aggressiveness in NSCLC. Statement of SignificanceWe performed a Gene Program Association Study (GPAS) to create an atlas of gene modules in cancer cells from NSCLCs. This atlas revealed extensive lineage vacillation, combinations of modules that defined late-stage disease, novel routes of lineage plasticity, and novel subtypes in lung adenocarcinoma or lung squamous cell carcinoma.

bioinformatics↗

bioRxiv2025

Epithelial miR-149-5p up-regulation is associated with immune evasion in progressive bronchial premalignant lesions

The molecular drivers bronchial premalignant lesion progression to invasive lung squamous cell carcinoma are not well defined. Prior work profiling longitudinally collected bronchial premalignant lesion biopsies by RNA sequencing defined a proliferative subtype, enriched with bronchial dysplasia. We found that a gene co-expression module associated with interferon gamma signaling and antigen processing/presentation was down-regulated in progressive/persistent versus regressive lesions within the proliferative subtype, suggesting a functional impact of these genes on immune evasion. RNA from these same premalignant lesions was profiled by microRNA (miRNA) sequencing and a miRNA-gene network analysis identified hsa-miR-149-5p as a potential regulator of this antigen presentation gene co-expression module associated with lesion progression. hsa-miR-149-5p was found to be predominantly expressed in the epithelium and up-regulated in progressive/persistent versus regressive proliferative lesions while targets of this miRNA, the transcriptional coactivator of MHC-I gene expression, NLRC5, and the genes it regulates were down-regulated. MicroRNA in situ hybridization of hsa-miR-149-5p in tissue from adjacent fixed biopsies showed that hsa-miR-149-5p was increased in areas of bronchial dysplasia in progressive/persistent versus regressive lesions. Imaging mass cytometry showed that NLRC5 protein expression was decreased in progressive/persistent versus regressive lesions within areas of hyperplasia, metaplasia, and dysplasia. Additionally, basal cells with high versus low levels of NLRC5 were found to be in close spatial proximity to CD8 T cells, suggesting that these cells exhibit increased functional MHC-I gene expression in lesions with low hsa-miR-149-5p expression. Collectively, our data suggests a functional role for hsa-miR-149-5p in bronchial premalignant lesions and may serve as a therapeutic target for PML immunomodulation. STATEMENT OF SIGNIFICANCEIntegrative analysis across bronchial premalignant lesions has identified and localized a potential regulator of immune evasion in progressive/persistent lesions that could be a novel therapeutic target.

cancer biology↗

bioRxiv2024

A human cytomegalovirus prefusion-like glycoprotein B subunit vaccine elicits similar humoral immunity to that of postfusion gB in mice

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects. Despite the global disease burden, there is no FDA-approved HCMV vaccine. The most efficacious HCMV vaccine candidates to date have used glycoprotein B (gB), a class III viral fusion protein, in its postfusion form. While some viral fusion proteins have been shown to elicit stronger neutralizing responses in their prefusion conformation, HCMV prefusion-like and postfusion gB were recently shown to elicit antibodies with similar fibroblast neutralization titers in mice. We aimed to define and compare the specificity and functionality of plasma IgG elicited by distinct prefusion-like and postfusion gB constructs. Prefusion-like and postfusion gB elicited comparable IgG responses that predominantly mapped to AD-5 antigenic domain known to elicit neutralizing antibodies. Interestingly, postfusion gB elicited significantly higher plasma IgG binding to cell-associated gB and antibody dependent cellular phagocytosis than that of prefusion-like gB. The vaccines elicited comparable neutralization titers of heterologous HCMV strain AD169r in fibroblasts, yet neither elicited neutralizing titers against the vaccine-matched strain Towne in fibroblasts. Our data indicate that gB in this prefusion-like conformation elicits similar specificity and functional humoral immunity to that of postfusion gB, unlike certain class I viral fusion proteins which have been used as vaccine antigens. These findings deepen our understanding of the immune response elicited by class III fusion proteins and may inform further design and testing of conformationally-dependent herpesvirus glycoprotein vaccine candidates.

immunology↗

bioRxiv2024

Neutrophil transcriptome diverges into two discrete trajectories in a murine model of severe Streptococcus pneumoniae pneumonia

Neutrophils were once considered a homogenous population of transcriptionally static, pathogen-killing cells, however, recent models have demonstrated neutrophil functional and transcriptional plasticity. We performed transcriptomic analyses in a murine model of pneumococcal pneumonia to investigate neutrophil plasticity and demonstrate that neutrophils are highly dynamic, leading to three distinct alveolar neutrophil populations - one immature (early bronchoalveolar lavage neutrophils [BALN]) and two mature (late BALN). Early BALNs produce high levels of inflammatory cytokine transcripts, maturing into late BALNs, including a pro-degranulation and phagocytosis population (late-degranulating BALN) or a population specializing in translation machinery and inflammatory cytokine production (late-cytokine producing BALN). Neutrophil metabolism is also regulated in a stepwise manner - tricarboxylic acid (TCA) cycle and respiratory electron transport chain (ETC) genes are downregulated as neutrophils migrate from the vasculature to the interstitium, lipid and carbohydrate metabolism genes are downregulated during migration from interstitium to the airspace. These transitions may be regulated by aspects of the integrated stress response (ISR), as key regulators including Eif2ak2 are upregulated in interstitial neutrophils. Overall, we demonstrate that pneumonic neutrophils are transcriptionally plastic, developing through two distinct transcriptional phenotypes in the airspace, and are metabolically and transcriptionally rewired with potential points of regulation occurring in the interstitial space.

immunology↗

bioRxiv2024

Repetitive head impacts induce neuronal loss and neuroinflammation in young athletes

Repetitive head impacts (RHI) sustained from contact sports are the largest risk factor for chronic traumatic encephalopathy (CTE). Currently, CTE can only be diagnosed after death and the multicellular cascade of events that trigger initial hyperphosphorylated tau (p-tau) deposition remain unclear. Further, the symptoms endorsed by young individuals with early disease are not fully explained by the extent of p-tau deposition, severely hampering development of therapeutic interventions. Here, we show that RHI exposure associates with a multicellular response in young individuals (<51 years old) prior to the onset of CTE p-tau pathology that correlates with number of years of RHI exposure. Leveraging single nucleus RNA sequencing of tissue from 8 control, 9 RHI-exposed, and 11 low stage CTE individuals, we identify SPP1+ inflammatory microglia, angiogenic and inflamed endothelial cell profiles, reactive astrocytes, and altered synaptic gene expression in excitatory and inhibitory neurons in all individuals with exposure to RHI. Surprisingly, we also observe a significant loss of cortical sulcus layer 2/3 neurons in contact sport athletes compared to controls independent of p-tau pathology. Finally, we identify TGFB1 as a potential signal mediating microglia-endothelial cell cross talk through ligand-receptor analysis. These results provide robust evidence that multiple years of RHI exposure is sufficient to induce lasting cellular alterations that may underlie p-tau deposition and help explain the early pathogenesis in young former contact sport athletes. Furthermore, these data identify specific cellular responses to repetitive head impacts that may direct future identification of diagnostic and therapeutic strategies for CTE.

neuroscience↗

bioRxiv2024

Multi-modal comparison of molecular programs driving nurse cell death and clearance in Drosophila melanogaster oogenesis

The death and clearance of nurse cells is a consequential milestone in Drosophila melanogaster oogenesis. In preparation for oviposition, the germline-derived nurse cells bequeath to the developing oocyte all their cytoplasmic contents and undergo programmed cell death. The death of the nurse cells is controlled non-autonomously and is precipitated by epithelial follicle cells of somatic origin acquiring a squamous morphology and acidifying the nurse cells externally. Alternatively, stressors such as starvation can induce the death of nurse cells earlier in mid-oogenesis, manifesting apoptosis signatures, followed by their engulfment by epithelial follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, both mediated by follicle cells, we compared their genome-wide transcriptional, translational, and secretion profiles before and after differentiating to acquire a phagocytic capability, as well as during well-fed and nutrient-deprived conditions. By coupling the GAL4-UAS system to Translating Ribosome Affinity Purification (TRAP-seq) and proximity labeling (HRP-KDEL) followed by Liquid Chromatography tandem mass-spectrometry, we performed high-throughput screens to identify pathways selectively activated or repressed by follicle cells to employ nurse cell-clearance routines contextually and preferentially. We also integrated two publicly available single-cell RNAseq atlases of the Drosophila ovary to define the transcriptomic profiles of follicle cells. In this report, we describe the genes and major pathways identified in the screens and the striking consequences to Drosophila melanogaster oogenesis caused by RNAi perturbation of prioritized candidates. To the best of our knowledge, our study is the first of its kind to comprehensively characterize two distinct apoptotic and non-apoptotic cell death paradigms in the same multi-cellular system. Beyond molecular differences in cell death, our investigation may also provide insights into how key systemic trade-offs are made between survival and reproduction when faced with physiological stress.

genetics↗

bioRxiv2024

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled Ifn{beta} superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

immunology↗

bioRxiv2024

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 [A] resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses. TeaserA structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.

molecular biology↗

bioRxiv2023

Matrix and analysis metadata standards (MAMS) to facilitate harmonization and reproducibility of single-cell data

A large number of genomic and imaging datasets are being produced by consortia that seek to characterize healthy and disease tissues at single-cell resolution. While much effort has been devoted to capturing information related to biospecimen information and experimental procedures, the metadata standards that describe data matrices and the analysis workflows that produced them are relatively lacking. Detailed metadata schema related to data analysis are needed to facilitate sharing and interoperability across groups and to promote data provenance for reproducibility. To address this need, we developed the Matrix and Analysis Metadata Standards (MAMS) to serve as a resource for data coordinating centers and tool developers. We first curated several simple and complex "use cases" to characterize the types of featureobservation matrices (FOMs), annotations, and analysis metadata produced in different workflows. Based on these use cases, metadata fields were defined to describe the data contained within each matrix including those related to processing, modality, and subsets. Suggested terms were created for the majority of fields to aid in harmonization of metadata terms across groups. Additional provenance metadata fields were also defined to describe the software and workflows that produced each FOM. Finally, we developed a simple listlike schema that can be used to store MAMS information and implemented in multiple formats. Overall, MAMS can be used as a guide to harmonize analysis-related metadata which will ultimately facilitate integration of datasets across tools and consortia. MAMS specifications, use cases, and examples can be found at https://github.com/single-cell-mams/mams/.

bioinformatics↗

bioRxiv2023

Decontamination of ambient and margin noise in droplet-based single cell protein expression data with DecontPro

Assays such as CITE-seq can measure the abundance of cell surface proteins on individual cells using antibody derived tags (ADTs). However, many ADTs have high levels of background noise that can obfuscate down-stream analyses. Using an exploratory analysis of PBMC datasets, we find that some droplets that were originally called "empty" due to low levels of RNA contained high levels of ADTs and likely corresponded to neutrophils. We identified a novel type of artifact in the empty droplets called a "spongelet" which has medium levels of ADT expression and is distinct from ambient noise. ADT expression levels in the spongelets correlate to ADT expression levels in the background peak of true cells in several datasets suggesting that they can contribute to background noise along with ambient ADTs. We then developed DecontPro, a novel Bayesian hierarchical model that can decontaminate ADT data by estimating and removing contamination from these sources. DecontPro outperforms other decontamination tools in removing aberrantly expressed ADTs while retaining native ADTs and in improving clustering specificity. Overall, these results suggest that identification of empty drops should be performed separately for RNA and ADT data and that DecontPro can be incorporated into CITE-seq workflows to improve the quality of downstream analyses.

bioinformatics↗

bioRxiv2022

A contamination focused approach for optimizing thesingle-cell RNA-seq experiment

Achieving high data quality in single-cell RNA-seq (scRNA-seq) experiments has always been a significant challenge stemming from minute signal that can be detected in individual cells. Droplet-based scRNA-seq additionally suffers from ambient contamination, comprising nucleic acid materials released by dead cells into the loading buffer and co-encapsulated with real cells, which further washes out real biological signals. Here, we developed quantitative, ambient contamination-based metrics and an associated software package that can both evaluate current datasets and guide new experimental optimizations. We performed a series of experimental optimizations using the inDrops platform to address the mechanical and microfluidic cell encapsulation aspect of an scRNA-seq experiment, with a focus on minimizing ambient contamination. We report improvements that can be achieved via cell fixation, microfluidic loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide insights into previously obscured factors that can affect scRNA-seq data quality and suggest mitigation strategies that can guide future experiments.

genomics↗