Jin Xu

Single-cell RNA sequencing measures gene expression in individual cells, but RNA released from damaged cells can be captured alongside RNA from intact cells, producing spurious signals that affect cell identification and biological interpretation. Although many computational tools aim to remove ambient RNA, their relative performance has not been systematically evaluated. Using simulated and experimental datasets from diverse tissues and species, we assessed seven tools for accuracy in estimating contamination levels, robustness across biological and technical conditions, and sensitivity to subtype-specific contamination. Here we show that the tools have distinct strengths, underscoring the need to match methods to data characteristics and research goals. DecontX provides the most accurate contamination-level estimation. scAR is the most robust across conditions but tends to overestimate contamination, whereas CellClear performs best for resolving closely related cell subtypes. This benchmark guides method selection to improve the reliability of single-cell analyses and identifies priorities for future method development.

Olfactory sensory neurons comprise a population of highly heterogeneous subtypes. One important mechanism contributing to neuronal diversity is post-transcriptional regulation, in which isoform heterogeneity is an important component. Comprehensive profiling of transcript isoforms in olfactory sensory neurons is therefore essential for understanding the molecular mechanisms underlying their diversity. By integrating single-cell long-read sequencing with conventional 3’ RNA sequencing, we demonstrated that these neurons contain extensive isoform diversity. We identified widespread UTR remodeling and isoform switching associated with developmental stage and spatial projection pattern, providing compelling evidence for the crucial regulatory mechanism underlying OSN diversity. Furthermore, we observed co-expression of multiple isoforms from the same olfactory receptor gene within individual olfactory sensory neurons, including some predicted to encode complete transmembrane domains. Together, these data provide a resource and framework for studying how specific isoforms contribute to olfactory sensory neuron identity, axon targeting, and the maintenance of singular olfactory receptor expression.

The restricted regenerative potential of adult hearts poses a significant barrier to effective repair following injury. In contrast to numerous vertebrates, mammalian hearts exhibit only transient neonatal renewal capacity during the initial days of life. Beyond cardiomyocytes, understanding the diverse compositions of non-cardiomyocytes (non-CMs) is imperative for maintaining heart microenvironment homeostasis during neonatal heart regeneration. Here, we conduct single-cell ATAC sequencing on neonatal hearts at varying time points post-apical resection to profile the epigenetic landscape. Intriguingly, fibroblasts and endothelial cells, as the most abundant populations in the heart, exhibit the most dynamic chromatin remodeling upon injury. Furthermore, we reveal CEBPD and AP-1 family transcriptional factors as pivotal trans-regulators orchestrating these alterations, governing beneficial fibroblast activation and endothelial cell angiogenesis crucial for cardiac regeneration, respectively. Collectively, our study delineates the cellular identity of non-CMs at the epigenome level using single-cell approaches, offering insights into cell type-targeted interventions for heart regeneration.

Objective: Single-cell transcriptome sequencing is a powerful tool for investigating cellular diversity in normal development and disease. However, prevalent methods predominantly employ 3′-end sequencing of transcripts, limiting the analysis of alternative splicing and other post-transcriptional processes. While full-length single-cell transcriptome sequencing methods, such as Smart-seq, offer more comprehensive information, but are restricted by low-throughput. To overcome these limitations, we propose a strategy that combines in situ reverse transcription and transposition with a high-throughput micro-fluid platform to enable scalable full-length transcriptome profiling at single-cell resolution.Methods: In this study, we utilized methanol fixation on cultured cells to evaluate RNA integrity and cellular preservation post-fixation. In situ reverse transcription followed by RNA/DNA hybrids transposition was performed to test the efficiency of these reactions. The transposed fragments were sequenced and investigated to determine transcriptome capture efficiency. Finally, we combined in situ reactions with the 10X Genomics scATAC-seq platform to prepare a single-cell transcriptome library, aiming to assess the feasibility of full-length transcriptome sequencing at the single-cell level using this combined approach.Results: Methanol fixation enables preservation of RNA and facilitates in situ reverse transcription of full-length cDNA. Importantly, cells maintain their integrality after reverse transcription and transposition even under low concentration of methanol. Reducing the methanol concentration to 40% further enhances transcript capture efficiency. At the single-cell level, this strategy enables the capture of full-length transcriptomes, demonstrating a great potential for application in single-cell sequencing.

Olfaction is crucial for animals' survival and adaptation. Unlike the strict singular expression of odorant receptor (OR) genes in vertebrate olfactory sensory neurons (OSNs), insects exhibit complex OR gene expression patterns. In honeybees (Apis mellifera), a significant expansion of OR genes implies a selection preference for the olfactory demands of social insects. However, the mechanisms underlying receptor expression specificity and their contribution to OSN divergence remain unclear. In this study, we used single-nucleus multiomics profiling to investigate the transcriptional regulation of OR genes and the cellular identity of OSNs in A. mellifera. We identified three distinct OR expression patterns, singular OR expression, co-expression of multiple OR genes with a single active promoter, and co-expression of multiple OR genes with multiple active promoters. Notably, ∼50% of OSNs co-expressed multiple OR genes, driven by polycistronic transcription of tandemly duplicated OR genes via a single active promoter. In these OSNs, their identity was determined by the first transcribed receptor. The divergent activation of the promoter for duplicated OR genes ensures the coordinated increased divergence of OSN population. By integrating multiomics data with genomic architecture, we illustrate how fundamental genetic mechanisms drive OR gene expansion and influence flanking regulatory elements, ultimately contributing to the cellular divergence of OSNs. Our findings highlight the interplay between gene duplication and regulatory evolution in shaping OSN diversity, providing new insights into the evolution and adaptation of olfaction in social insects. This study also sheds light on how genetic innovations contribute to the evolution of complex traits.

Mitochondrial DNA (mtDNA) variants hold promise as endogenous barcodes for tracking human cell lineages, but their efficacy as reliable lineage markers are hindered by the complex dynamics of mtDNA in somatic tissues. Here, we use computational modeling and single-cell genomics to thoroughly interrogate the origin and clonal dynamics of mtDNA variants across various biological settings. Our findings reveal that the majority of mtDNA variants which are specifically present in a cell subpopulation, termed subpopulation-specific variants, are pre-existing heteroplasmies in the first cell instead of de novo somatic mutations during divisions. Moreover, subpopulation-specific variants demonstrate limited discriminatory power among different genuine lineages under weak clonal expansion; however, certain subpopulation-specific variants with consistently high frequencies among a subpopulation are capable of faithfully labeling cell lineages in scenarios of stringent clonal expansion, such as strongly expanded T cell populations in diseased conditions and clonal hematopoiesis in aged individuals. Inspired by our simulations, we introduce a lineage informative score, facilitating the identification of reliable mitochondrial lineage tracing markers across different modalities of single-cell genomic data. Combining computational modeling and single-cell sequencing, our study reveals that the performance of mitochondrial lineage tracing is highly dependent on the extent of clonal expansion, which thus should be considered when applying mitochondrial lineage tracing.

Background: Anti‐N‐methyl‐D‐aspartate receptor encephalitis (anti‐NMDARE) is a prevalent type of autoimmune encephalitis caused by antibodies targeting the NMDAR's GluN1 subunit. While significant progress has been made in elucidating the pathophysiology of autoimmune diseases, the immunological mechanisms underlying anti‐NMDARE remain elusive. This study aimed to characterize immune cell interactions and dysregulation in anti‐NMDARE by leveraging single‐cell multi‐omics sequencing technologies.Methods: Peripheral blood mononuclear cells (PBMCs) from patients in the acute phase of anti‐NMDARE and healthy controls were sequenced using single‐cell joint profiling of transcriptome and chromatin accessibility. Differential gene expression analysis, transcription factor activity profiling, and cell‐cell communication modeling were performed to elucidate the immune mechanisms underlying the disease. In parallel, single‐cell B cell receptor sequencing (scBCR‐seq) and repertoire analysis were conducted to assess antigen‐driven clonal expansion within the B cell population.Results: The study revealed a significant clonal expansion of B cells, particularly plasma cells, in anti‐NMDARE patients. The novel finding of type I interferon (IFN‐I) pathway activation suggests a regulatory mechanism that may drive this expansion and enhance antibody secretion. Additionally, activation of Toll‐like receptor 2 (TLR2) in myeloid cells was noted, which may connect to tumor necrosis factor‐alpha (TNF‐α) secretion. This cytokine may contribute to the activation of B and T cells, thereby perpetuating immune dysregulation.Conclusions: This study presents a comprehensive single‐cell multi‐omics characterization of immune dysregulation in anti‐NMDARE, highlighting the expansion of B cell and the activation of the IFN‐I and TLR2 pathways. These findings provide deeper insights into the molecular mechanism driving the pathogenesis of anti‐NMDARE and offer promising targets for future therapeutic intervention.Key points: Significant B cell clonal expansion, particularly in plasma cells, driven by antigen recognition. IFN‐I pathway activation in plasma cells boosts their antibody production and potentially exacerbates immune dysregulation. TLR2 pathway activation in myeloid cells contributes to TNF‐α secretion and could influence adaptive immune responses.

Multiplexing across donors has emerged as a popular strategy to increase throughput, reduce costs, overcome technical batch effects, and improve doublet detection in single-cell genomic studies. To eliminate additional experimental steps, endogenous nuclear genome variants are used for demultiplexing pooled single-cell RNA sequencing (scRNA-seq) data by several computational tools. However, these tools have limitations when applied to single-cell sequencing methods that do not cover nuclear genomic regions well, such as single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq). Here, we demonstrate that mitochondrial germline variants are an alternative, robust, and computationally efficient endogenous barcode for sample demultiplexing. We propose MitoSort, a tool that uses mitochondrial germline variants to assign cells to their donor of origin and identify cross-genotype doublets in single-cell genomics datasets. We evaluate its performance by using in silico pooled mitochondrial scATAC-seq (mtscATAC-seq) libraries and experimentally multiplexed data with cell hashtags. MitoSort achieves high accuracy and efficiency in genotype clustering and doublet detection for mtscATAC-seq data, addressing the limitations of current computational techniques tailored for scRNA-seq data. Moreover, MitoSort exhibits versatility and can be applied to various single-cell sequencing approaches beyond mtscATAC-seq, provided the mitochondrial variants are reliably detected. Furthermore, we demonstrate the application of MitoSort in a case study where B cells from eight donors were pooled and assayed by single-cell multi-omics sequencing. Altogether, our results demonstrate the accuracy and efficiency of MitoSort, which enables reliable sample demultiplexing in various single-cell genomic applications. MitoSort is available at https://github.com/tangzhj/MitoSort.

In adult gonads, disruption of somatic sexual identity leads to defective gametogenesis and infertility. However, the underlying mechanisms by which somatic signals regulate germline cells to achieve proper gametogenesis remain unclear. In our previous study, we introduced the chinmo^{Sex Transformation} (chinmo^ST) mutant Drosophila testis phenotype as a valuable model for investigating the mechanisms underlying sex maintenance. In chinmo^ST testes, depletion of the Janus Kinase-Signal Transducer and Activator of Transcription downstream effector Chinmo from somatic cyst stem cells (CySCs) feminizes somatic cyst cells and arrests germline differentiation. Here, we use single-cell RNA sequencing to uncover chinmo^ST-specific cell populations and their transcriptomic changes during sex transformation. Comparative analysis of intercellular communication networks between wild-type and chinmo^ST testes revealed disruptions in several soma-germline signaling pathways in chinmo^ST testes. Notably, the insulin signaling pathway exhibited significant enhancement in germline stem cells (GSCs). Chinmo cleavage under targets and tagmentation (CUT&Tag) assay revealed that Chinmo directly regulates two male sex determination factors, doublesex (dsx) and fruitless (fru), as well as Ecdysone-inducible gene L2 (ImpL2), a negative regulator of the insulin signaling pathway. Further genetic manipulations confirmed that the impaired gametogenesis observed in chinmo^ST testes was partly contributed by dysregulation of the insulin signaling pathway. In summary, our study demonstrates that somatic sex maintenance promotes normal spermatogenesis through Chinmo-mediated conserved sex determination and the insulin signaling pathway. Our work offers new insights into the complex mechanisms of somatic stem cell sex maintenance and soma-germline communication at the single-cell level. Additionally, our discoveries highlight the potential significance of stem cell sex instability as a novel mechanism contributing to testicular tumorigenesis.