Matthew Ritchie

• PhD, University of Melbourne
• The Walter and Eliza Hall Institute of Medical Research

RNA sequencing (RNA-seq) is widely used in biomedical research, advancing our understanding of gene expression across biological systems. Traditional methods require upstream RNA extraction from biological inputs, adding time and expense to workflows. We developed TIRE-seq (Turbocapture Integrated RNA Expression Sequencing) to address these challen...

The D4Z4 locus is a macrosatellite array on chromosome 4q that normally comprises 8 to >100 3.3-kb repeat units. Its size and repetitiveness render it refractory to most sequencing technologies; consequently its genetic and epigenetic architectures remain incompletely understood despite their relevance to human health, in particular facioscapulohum...

Spatial transcriptomics technology has developed rapidly in recent years, with various sequencing-based platforms such as 10x Visium, Slide-seq and Stereo-seq becoming widely used by researchers. Each platform brings its own set of protocols and customised data analysis pipelines which presents challenges when the goal is to obtain uniformly prepro...

Background Spatial transcriptomics allows gene expression to be measured within complex tissue contexts. Among the array of spatial capture technologies available is 10x Genomics’ Visium platform, a popular method which enables transcriptome-wide profiling of tissue sections. Visium offers a range of sample handling and library construction methods...

Long-read sequencing technologies have transformed the field of epigenetics by enabling direct, single-base resolution detection of DNA modifications, such as methylation. This produces novel opportunities for studying the role of DNA methylation in gene regulation, imprinting, and disease. However, the unique characteristics of long-read data, inc...

Long-read RNA sequencing has significantly advanced transcriptomics by enabling the full length of transcripts to be assessed. However, current analysis methods often depend on a high-quality reference genome and gene annotation. Recently, de novo assembly methods have been developed to utilise long-read data in cases where a reference genome is un...

Single-cell long-read sequencing has transformed our understanding of isoform usage and the mutation heterogeneity between cells. Despite unbiased in-depth analysis, the low sequencing throughput often results in insufficient read coverage thereby limiting our ability to perform mutation calling for specific genes. Here, we developed a single-cell...

RNA sequencing is widely used in biomedical research, advancing our understanding of gene expression across biological systems. Traditional methods require upstream RNA extraction from biological inputs, adding time and expense to workflows. We developed TIRE-seq (Turbocapture Integrated RNA Expression Sequencing) to address these challenges. TIRE-...

Venetoclax, a first-in-class BH3 mimetic drug targeting BCL-2, has improved outcomes for patients with chronic lymphocytic leukemia (CLL). Early measurements of the depth of the venetoclax treatment response, assessed by minimal residual disease, are strong predictors of long-term clinical outcomes. Yet, there are limited data concerning the early...

Despite recent advances made toward improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical...

Long-read sequencing technologies have transformed the field of epigenetics by enabling direct, single-base resolution detection of DNA modifications, such as methylation. This produces novel opportunities for studying the role of DNA methylation in gene regulation, imprinting, and disease. However, the unique characteristics of long-read data, inc...

Single-cell RNA sequencing (scRNA-seq) is a powerful technology that enables the measurement of gene expression in individual cells. Such precision provides insights into cellular heterogeneity that bulk methods might overlook. Fragile cells, in particular neutrophils, have posed significant challenges for scRNA-Seq due to their ex vivo fragility,...

X-linked genetic disorders typically affect females less severely than males due to the presence of a second X Chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X-Chromosome inactivation. Accurate measurement of X inactivation skew is cruci...

Germination involves highly dynamic transcriptional programs as the cells of seeds reactivate and express the functions necessary for establishment in the environment. Individual cell types have distinct roles within the embryo, so must therefore have cell type-specific gene expression and gene regulatory networks. We can better understand how the...

Recent developments of sequencing-based spatial transcriptomics (sST) have catalyzed important advancements by facilitating transcriptome-scale spatial gene expression measurement. Despite this progress, efforts to comprehensively benchmark different platforms are currently lacking. The extant variability across technologies and datasets poses chal...

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust p...

X-linked genetic disorders typically affect females less severely than males due to the presence of a second X chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X chromosome inactivation. Accurate measurement of X inactivation skew is cruci...

Venetoclax, a first-in-class BH3 mimetic drug targeting BCL-2, has improved outcomes for patients with chronic lymphocytic leukemia (CLL). Early measurements of the depth of the venetoclax treatment response, assessed by minimal residual disease, are strong predictors of long-term clinical outcomes. Yet, there are limited data concerning the early...

Motivation The process of analyzing high throughput sequencing data often requires the identification and extraction of specific target sequences. This could include tasks such as identifying cellular barcodes and UMIs in single cell data, and specific genetic variants for genotyping. However, existing tools which perform these functions are often...

Single-cell long-read sequencing has transformed our understanding of isoform usage and the mutation heterogeneity between cells. Despite unbiased in-depth analysis, the low sequencing throughput often results in insufficient read coverage thereby limiting our ability to perform mutation calling for specific genes. Here, we developed a s ingle- c e...

In transcriptomic analyses, it is helpful to keep track of the strand of the RNA molecules. However, the Oxford Nanopore long-read cDNA sequencing protocols generate reads that correspond to either the first or second-strand cDNA, therefore the strandedness of the initial transcript has to be inferred bioinformatically. Reverse transcription and PC...

Differential expression analysis of RNA-seq is one of the most commonly performed bioinformatics analyses. Transcript-level quantifications are inherently more uncertain than gene-level read counts because of ambiguous assignment of sequence reads to transcripts. While sequence reads can usually be assigned unambiguously to a gene, reads are very o...

Recent advancements of sequencing-based spatial transcriptomics (sST) have catalyzed significant advancements by facilitating transcriptome-scale spatial gene expression measurement. Despite this progress, efforts to comprehensively benchmark different platforms are currently lacking. The extant variability across technologies and datasets poses ch...

Despite recent advances made towards improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially non-functional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinica...

scPipe is a flexible R/Bioconductor package originally developed to analyse platform-independent single-cell RNA-Seq data. To expand its preprocessing capability to accommodate new single-cell technologies, we further developed scPipe to handle single-cell ATAC-Seq and multi-modal (RNA-Seq and ATAC-Seq) data. After executing multiple data cleaning...

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust p...

The lack of benchmark data sets with inbuilt ground-truth makes it challenging to compare the performance of existing long-read isoform detection and differential expression analysis workflows. Here, we present a benchmark experiment using two human lung adenocarcinoma cell lines that were each profiled in triplicate together with synthetic, splice...

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associat...

scPipe is a flexible R/Bioconductor package originally developed to analyse platform-independent single-cell RNA-Seq data. To expand its preprocessing capability to accommodate new single-cell technologies, we further developed scPipe to handle single-cell ATAC-Seq and multi-modal (RNA-Seq and ATAC-Seq) data. After executing multiple data cleaning...

Skeletal muscle contains a resident population of somatic stem cells capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use metabolomics and mass spectrometry imaging (MSI) to identity a state of localized hyperglycaemia following skeletal muscle injury. We sh...

Group heteroscedasticity is commonly observed in pseudo-bulk single-cell RNA-seq datasets and its presence can hamper the detection of differentially expressed genes. Since most bulk RNA-seq methods assume equal group variances, we introduce two new approaches that account for heteroscedastic groups, namely voomByGroup and voomWithQualityWeights us...

A major challenge in the analysis of RNA-seq data at the transcript-level is accounting for the variability introduced during quantification of RNA sequencing reads. This variability is due to the high levels of sequence similarity among transcripts annotated to the same genomic locus and the mapping ambiguity resulting from the assignment of seque...

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methy...

Venetoclax is an effective treatment for certain blood cancers, such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). However, most patients relapse while on venetoclax and further treatment options become limited. Combining venetoclax with immunotherapies is an attractive approach; however, a detailed understanding of how ve...

Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mech...

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methy...

Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both...

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B–related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through ho...

Group heteroscedasticity is commonly observed in pseudo-bulk single-cell RNA-seq datasets and when not modelled appropriately, its presence can hamper the detection of differentially expressed genes. Most bulk RNA-seq methods assume equal group variances which will under- and/or over-estimate the true variability in such datasets. We present two me...

The current lack of benchmark datasets with inbuilt ground-truth makes it challenging to compare the performance of existing long-read isoform detection and differential expression analysis workflows. Here, we present a benchmark experiment using two human lung adenocarcinoma cell lines that were each profiled in triplicate together with synthetic,...

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes...

Venetoclax inhibits the pro-survival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL, but with eventual loss of efficacy. A spectrum of sub-clonal genetic changes associated with venetoclax resistance h...

[This corrects the article DOI: 10.1016/j.dib.2022.107828.].

Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both...

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensatio...

Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mech...

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline ( FLAMES ) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved funct...

Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryo...

Glimma 1.0 introduced intuitive, point-and-click interactive graphics for differential gene expression analysis. Here, we present a major update to Glimma that brings improved interactivity and reproducibility using high-level visualization frameworks for R and JavaScript. Glimma 2.0 plots are now readily embeddable in R Markdown, thus allowing use...

Vagal sensory neurons contribute to the symptoms and pathogenesis of inflammatory pulmonary diseases through processes that involve changes to their morphological and functional characteristics. The alarmin high mobility group box-1 (HMGB1) is an early mediator of pulmonary inflammation and can have actions on neurons in a range of inflammatory set...

Glimma 1.0 introduced intuitive, point-and-click interactive graphics for differential gene expression analysis. Here, we present a major update to Glimma which brings improved interactivity and reproducibility using high-level visualisation frameworks for R and JavaScript. Glimma 2.0 plots are now readily embeddable in R Markdown, thus allowing us...

Despite advances in single cell multi-omics, a single stem or progenitor cell can only be tested once. We developed ‘clonal multi-omics’, where daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel ‘SISter’ assays are examined for either gene expressi...

While the intrinsic apoptosis pathway is thought to play a central role in shaping the B cell lineage, its precise role in mature B cell homeostasis remains elusive. Using mice in which mature B cells are unable to undergo apoptotic cell death, we show that apoptosis constrains follicular B (FoB) cell lifespan but plays no role in marginal zone B (...

Single-cell RNA sequencing (scRNA-seq) technologies and associated analysis methods have undergone rapid development in recent years. This includes methods for data preprocessing, which assign sequencing reads to genes to create count matrices for downstream analysis. Several packaged preprocessing workflows have been developed that aim to provide...

Development of a branching tree in the embryonic lung is critical for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen and identified Aurora kinase b (Aurkb) as a critica...

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets and at the facioscapulohumeral muscular dystrophy associated macro-array, D4Z4. Heightened SMCHD1 silen...

Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two na...

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel “sister” assays are examined either for gene express...

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. Ho...

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that a...

Background: The data produced by long-read third-generation sequencers have unique characteristics compared to short-read sequencing data, often requiring tailored analysis tools for tasks ranging from quality control to downstream processing. The rapid growth in software that addresses these challenges for different genomics applications is diffi...

Motivation A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. Tools for effective visualization of data generated by this platform to assess changes in methylation profiles between samples from different experimental groups remains a challenge. Results To make visualizat...

Differential expression analysis of genomic data types, such as RNA-sequencing experiments, use linear models to determine the size and direction of the changes in gene expression. For RNA-sequencing, there are several established software packages for this purpose accompanied with analysis pipelines that are well described. However, there are two...

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin⁻c-Kit⁺Sca-1⁺Flt3⁺ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cel...

Genomic imprinting establishes parental allele-biased expression of a suite of mammalian genes based on parent-of-origin specific epigenetic marks. These marks are under the control of maternal effect proteins supplied in the oocyte. Here we report epigenetic repressor Smchd1 as a novel maternal effect gene that regulates the imprinted expression o...

RNA-seq datasets can contain millions of intron reads per library that are typically removed from downstream analysis. Only reads overlapping annotated exons are considered to be informative since mature mRNA is assumed to be the major component sequenced, especially for poly(A) RNA libraries. In this study, we show that intron reads are informativ...

Alternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues...

Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to small library sizes and high sequence error, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopo...

Extrinsic regulation of single haematopoietic stem and progenitor cell (HSPC) fate is crucial for immune cell development. Here, we examine the aetiology of Flt3 ligand (Flt3L)-mediated emergency development of type 1 conventional dendritic cells (cDC1s), which results in enhanced immunity against infections and cancer. Using cellular barcoding, we...

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archet...

Modulators of epithelial to mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human...

Introduction Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer and though most patients initially respond to platinum-based chemotherapy, resistance rapidly develops. Immunotherapy has promise in the treatment of lung cancer, however SCLC patients exhibit poor overall responses highlighting the necessity for alternative ap...

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing–based transcriptomic analysis in mice and humans, we studied the changing transc...

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells,...

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of lon...

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary proce...

Genomic imprinting establishes allele-biased expression of a suite of mammalian genes based on their parent of origin. Imprinted expression is achieved via parent-of-origin specific epigenetic marks under the control of maternal effect proteins supplied in the oocyte. Here we report Structural maintenance of chromosomes hinge domain containing 1 (S...

Motivation: Bioinformatic analysis of f gene expression data is a rapidly evolving field. Hundreds of bespoke methods have been developed in the past few years to deal with various aspects of single-cell analysis and consensus on the most appropriate methods to use under different settings is still emerging. Benchmarking the many methods is theref...

The KRAS oncoprotein, a critical driver in 33% of lung adenocarcinoma (LUAD), has remained an elusive clinical target due to its perceived undruggable nature. The identification of dependencies borne through common co-occurring mutations are sought to more effectively target KRAS-mutant lung cancer. Approximately 20% of KRAS-mutant LUAD carry loss-...

Although female pluripotency significantly differs to male, complications with in vitro culture of female embryonic stem cells (ESC) have severely limited the use and study of these cells. We report a replenishable female ESC system, Xmas, that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ES...

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategie...

Dendritic cells (DCs) are immune cells important for the detection and immunity against pathogens, self-antigens and cancer. They include 3 subtypes, conventional DC type 1 (cDC1), cDC type 2 (cDC2) and plasmacytoid DC (pDC) which all derive from a common hematopoietic stem cell progenitor population (HSPCs). Recent evidence has shown that this pop...