ORCID Profile
0000-0002-7383-0609
Current Organisation
Walter and Eliza Hall Institute of Medical Research
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Bioinformatics | Genetics | Applied Statistics | Epigenetics (incl. Genome Methylation and Epigenomics) | Gene Expression (incl. Microarray and other genome-wide approaches) | Genome Structure and Regulation | Biochemistry and Cell Biology | Systems Biology | Cell Development, Proliferation and Death
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Mathematical Sciences |
Publisher: Springer Science and Business Media LLC
Date: 26-05-2010
Publisher: Cold Spring Harbor Laboratory
Date: 22-07-2022
DOI: 10.1101/2022.07.22.501076
Abstract: 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, spliced, spike-in RNAs (“sequins”). S les were deeply sequenced on both Illumina short-read and Oxford Nanopore Technologies long-read platforms. Alongside the ground-truth available via the sequins, we created in silico mixture s les to allow performance assessment in the absence of true positives or true negatives. Our results show that, StringTie2 and bambu outperformed other tools from the 6 isoform detection tools tested, DESeq2, edgeR and limma-voom were best amongst the 5 differential transcript expression tools tested and there was no clear front-runner for performing differential transcript usage analysis between the 5 tools compared, which suggests further methods development is needed for this application.
Publisher: F1000 Research Ltd
Date: 12-0003
DOI: 10.12688/F1000RESEARCH.27893.1
Abstract: 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 crucial steps in the analysis process that can be a stumbling block for many -- the set up an appropriate model via design matrices and the set up of comparisons of interest via contrast matrices. These steps are particularly troublesome because an extensive catalogue for design and contrast matrices does not currently exist. One would usually search for ex le case studies across different platforms and mix and match the advice from those sources to suit the dataset they have at hand. This article guides the reader through the basics of how to set up design and contrast matrices. We take a practical approach by providing code and graphical representation of each case study, starting with simpler ex les (e.g. models with a single explanatory variable) and move onto more complex ones (e.g. interaction models, mixed effects models, higher order time series and cyclical models). Although our work has been written specifically with a limma -style pipeline in mind, most of it is also applicable to other software packages for differential expression analysis, and the ideas covered can be adapted to data analysis of other high-throughput technologies. Where appropriate, we explain the interpretation and differences between models to aid readers in their own model choices. Unnecessary jargon and theory is omitted where possible so that our work is accessible to a wide audience of readers, from beginners to those with experience in genomics data analysis.
Publisher: Oxford University Press (OUP)
Date: 29-04-2015
DOI: 10.1093/NAR/GKV412
Publisher: Oxford University Press (OUP)
Date: 18-11-2009
DOI: 10.1093/NAR/GKP942
Publisher: Springer Science and Business Media LLC
Date: 30-01-2020
DOI: 10.1038/S41590-020-0606-8
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: Elsevier BV
Date: 07-2014
Publisher: Springer Science and Business Media LLC
Date: 11-11-2021
DOI: 10.1186/S13059-021-02525-6
Abstract: A modified Chromium 10x droplet-based protocol that subs les 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 functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on in idual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.
Publisher: Springer Science and Business Media LLC
Date: 03-2021
Publisher: Wiley
Date: 25-05-2016
DOI: 10.1111/BJH.14155
Abstract: Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime ex le. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.
Publisher: Wiley
Date: 03-2021
Publisher: Oxford University Press (OUP)
Date: 09-04-2021
Abstract: 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 nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (‘sequins’) as well as a mouse neural stem cell dataset from s les with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent s les. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.
Publisher: Oxford University Press (OUP)
Date: 29-01-2021
DOI: 10.1093/GIGASCIENCE/GIAB003
Abstract: 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 difficult to keep track of, which makes it hard for users to choose the most appropriate tool for their analysis goal and for developers to identify areas of need and existing solutions to benchmark against. We describe the implementation of long-read-tools.org, an open-source database that organizes the rapidly expanding collection of long-read data analysis tools and allows its exploration through interactive browsing and filtering. The current database release contains 478 tools across 32 categories. Most tools are developed in Python, and the most frequent analysis tasks include base calling, de novo assembly, error correction, quality checking/filtering, and isoform detection, while long-read single-cell data analysis and transcriptomics are areas with the fewest tools available. Continued growth in the application of long-read sequencing in genomics research positions the long-read-tools.org database as an essential resource that allows researchers to keep abreast of both established and emerging software to help guide the selection of the most relevant tool for their analysis needs.
Publisher: Springer Science and Business Media LLC
Date: 25-09-2023
Publisher: Springer Science and Business Media LLC
Date: 27-04-2020
DOI: 10.1038/S41418-020-0541-0
Abstract: 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, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER + ) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER + breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER + cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.
Publisher: Public Library of Science (PLoS)
Date: 31-10-2008
Publisher: Springer Science and Business Media LLC
Date: 12-2021
DOI: 10.1186/S13059-021-02552-3
Abstract: Single-cell RNA-sequencing (scRNA-seq) technologies and associated analysis methods have rapidly developed in recent years. This includes preprocessing methods, which assign sequencing reads to genes to create count matrices for downstream analysis. While several packaged preprocessing workflows have been developed to provide users with convenient tools for handling this process, how they compare to one another and how they influence downstream analysis have not been well studied. Here, we systematically benchmark the performance of 10 end-to-end preprocessing workflows ( Cell Ranger , Optimus , salmon alevin , alevin-fry , kallisto bustools , dropSeqPipe , scPipe , zUMIs , celseq2 , and scruff ) using datasets yielding different biological complexity levels generated by CEL-Seq2 and 10x Chromium platforms. We compare these workflows in terms of their quantification properties directly and their impact on normalization and clustering by evaluating the performance of different method combinations. While the scRNA-seq preprocessing workflows compared vary in their detection and quantification of genes across datasets, after downstream analysis with performant normalization and clustering methods, almost all combinations produce clustering results that agree well with the known cell type labels that provided the ground truth in our analysis. In summary, the choice of preprocessing method was found to be less important than other steps in the scRNA-seq analysis process. Our study comprehensively compares common scRNA-seq preprocessing workflows and summarizes their characteristics to guide workflow users.
Publisher: Cold Spring Harbor Laboratory
Date: 18-01-2021
DOI: 10.1101/2021.01.18.426757
Abstract: 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 s les from different experimental groups remains a challenge. To make visualization of methylation changes more straightforward, we developed the R/Bioconductor package NanoMethViz . Our software can handle methylation calls generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the s le-level, we use multidimensional scaling to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across in idual reads along the genome using the spaghetti plot, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at ackages/NanoMethViz .
Publisher: Oxford University Press (OUP)
Date: 28-11-2016
DOI: 10.1093/NAR/GKW1063
Publisher: American Society of Hematology
Date: 20-08-2020
Abstract: 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 acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
Publisher: Springer Science and Business Media LLC
Date: 11-11-2009
Abstract: Illumina Sentrix-6 Whole-Genome Expression BeadChips are relatively new microarray platforms which have been used in many microarray studies in the past few years. These Chips have a unique design in which each Chip contains six microarrays and each microarray consists of two separate physical strips, posing special challenges for precise between-array normalization of expression values. None of the normalization strategies proposed so far for this microarray platform allow for the possibility of systematic variation between the two strips comprising each array. That this variation can be substantial is illustrated by a data ex le. We demonstrate that normalizing at the strip-level rather than at the array-level can effectively remove this between-strip variation, improve the precision of gene expression measurements and discover more differentially expressed genes. The gain is substantial, yielding a 20% increase in statistical information and doubling the number of genes detected at a 5% false discovery rate. Functional analysis reveals that the extra genes found tend to have interesting biological meanings, dramatically strengthening the biological conclusions from the experiment. Strip-level normalization still outperforms array-level normalization when non-expressed probes are filtered out. Plots are proposed which demonstrate how the need for strip-level normalization relates to inconsistent intensity range variation between the strips. Strip-level normalization is recommended for the preprocessing of Illumina Sentrix-6 BeadChips whenever the intensity range is seen to be inconsistent between the strips. R code is provided to implement the recommended plots and normalization algorithms.
Publisher: F1000 Research Ltd
Date: 17-06-2016
DOI: 10.12688/F1000RESEARCH.9005.1
Abstract: The ability to easily and efficiently analyse RNA-sequencing data is a key strength of the Bioconductor project. Starting with counts summarised at the gene-level, a typical analysis involves pre-processing, exploratory data analysis, differential expression testing and pathway analysis with the results obtained informing future experiments and validation studies. In this workflow article, we analyse RNA-sequencing data from the mouse mammary gland, demonstrating use of the popular edgeR package to import, organise, filter and normalise the data, followed by the limma package with its voom method, linear modelling and empirical Bayes moderation to assess differential expression and perform gene set testing. This pipeline is further enhanced by the Glimma package which enables interactive exploration of the results so that in idual s les and genes can be examined by the user. The complete analysis offered by these three packages highlights the ease with which researchers can turn the raw counts from an RNA-sequencing experiment into biological insights using Bioconductor.
Publisher: F1000 Research Ltd
Date: 30-11-2016
DOI: 10.12688/F1000RESEARCH.9005.2
Abstract: The ability to easily and efficiently analyse RNA-sequencing data is a key strength of the Bioconductor project. Starting with counts summarised at the gene-level, a typical analysis involves pre-processing, exploratory data analysis, differential expression testing and pathway analysis with the results obtained informing future experiments and validation studies. In this workflow article, we analyse RNA-sequencing data from the mouse mammary gland, demonstrating use of the popular edgeR package to import, organise, filter and normalise the data, followed by the limma package with its voom method, linear modelling and empirical Bayes moderation to assess differential expression and perform gene set testing. This pipeline is further enhanced by the Glimma package which enables interactive exploration of the results so that in idual s les and genes can be examined by the user. The complete analysis offered by these three packages highlights the ease with which researchers can turn the raw counts from an RNA-sequencing experiment into biological insights using Bioconductor.
Publisher: Springer Science and Business Media LLC
Date: 13-09-2019
DOI: 10.1038/S41467-019-12164-Y
Abstract: 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-of-function mutations in KEAP1 , a negative regulator of the antioxidant response transcription factor NFE2L2/NRF2. We demonstrate that Keap1 -deficient Kras G12D lung tumors arise from a bronchiolar cell-of-origin, lacking pro-tumorigenic macrophages observed in tumors originating from alveolar cells. Keap1 loss activates the pentose phosphate pathway, inhibition of which, using 6-AN, abrogated tumor growth. These studies highlight alternative therapeutic approaches to specifically target this unique subset of KRAS -mutant LUAD cancers.
Publisher: Elsevier BV
Date: 09-2015
Publisher: Oxford University Press (OUP)
Date: 25-10-2008
DOI: 10.1093/BIOINFORMATICS/BTN557
Abstract: Summary: With their many replicates and their random layouts, Illumina BeadArrays provide greater scope fordetecting spatial artefacts than do other microarray technologies. They are also robust to artefact exclusion, yet there is a lack of tools that can perform these tasks for Illumina. We present BASH, a tool for this purpose. BASH adopts the concepts of Harshlight, but implements them in a manner that utilizes the unique characteristics of the Illumina technology. Using bead-level data, spatial artefacts of various kinds can thus be identified and excluded from further analyses. Availability: The beadarray Bioconductor package (version 1.10 onwards), www.bioconductor.org Contact: andy.lynch@cancer.org.uk Supplementary information: Additional information and a vignette are included in the beadarray package.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2017
DOI: 10.1038/IJO.2017.180
Abstract: Inflammatory factors derived from adipose tissue have been implicated in mediating insulin resistance in obesity. We sought to identify these using explanted human adipose tissue exposed to innate and adaptive immune stimuli. Subcutaneous and omental adipose tissue from obese, insulin-resistant donors was cultured in the presence of macrophage and T-cell stimuli, and the conditioned medium tested for its ability to inhibit insulin-stimulated glucose uptake into human Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes. The nature of the inhibitory factor in conditioned medium was characterized physicochemically, inferred by gene microarray analysis and confirmed by antibody neutralization. Conditioned medium from omental adipose tissue exposed to a combination of macrophage- and T-cell stimuli inhibited insulin action and adiponectin secretion in SGBS adipocytes. This effect was associated with a pronounced change in adipocyte morphology, characterized by a decreased number of lipid droplets of increased size. The bioactivity of conditioned medium was abolished by trypsin treatment and had a molecular weight of 46 kDa by gel filtration. SGBS adipocytes exposed to a bioactive medium expressed multiple gene transcripts regulated by interferon-gamma (IFN-γ). Recombinant human IFN-γ recapitulated the effects of the bioactive medium and neutralizing antibody against IFN-γ but not other candidate factors abrogated medium bioactivity. IFN-γ released from inflamed omental adipose tissue may contribute to the metabolic abnormalities seen in human obesity.
Publisher: Springer Science and Business Media LLC
Date: 07-12-2014
DOI: 10.1007/S00125-013-3129-0
Abstract: Obesity and dysglycaemia are major risk factors for type 2 diabetes. We determined if obese people undergoing laparoscopic adjustable gastric banding (LAGB) had a reduced risk of progressing from impaired fasting glucose (IFG) to diabetes. This was a retrospective cohort study of obese people with IFG who underwent LAGB. Weight and diabetes outcomes after a minimum follow-up period of 4 years (mean ± SD 6.1 ± 1.7 years) were compared with those of Australian adults with IFG from a population-based study (AusDiab). We identified 281 LAGB patients with baseline IFG. Their mean ± SD age and BMI were 46 ± 9 years and 46 ± 9 kg/m(2), respectively. The diabetes incidence for patients in the lowest, middle and highest weight loss tertile were 19.1, 3.4 and 1.8 cases/1,000 person-years, respectively. The AusDiab cohort had a lower BMI (28 ± 5 kg/m(2)) and a diabetes incidence of 12.5 cases/1,000 person-years. This increased to 20.5 cases/1,000 person-years when analysis was restricted to the 322 obese AusDiab participants, which was higher than the overall rate of 8.2 cases/1,000 person-years seen in the LAGB group (p = 0.02). Multivariable analysis of the combined LAGB and AusDiab data suggested that LAGB was associated with ∼75% lower risk of diabetes (OR 0.24 [95% CI 0.10, 0.57], p = 0.004). In obese people with IFG, weight loss after LAGB is associated with a substantially reduced risk of progressing to diabetes over ≥4 years. Bariatric surgery may be an effective diabetes prevention strategy in this population.
Publisher: F1000 Research Ltd
Date: 28-12-2018
DOI: 10.12688/F1000RESEARCH.9005.3
Abstract: The ability to easily and efficiently analyse RNA-sequencing data is a key strength of the Bioconductor project. Starting with counts summarised at the gene-level, a typical analysis involves pre-processing, exploratory data analysis, differential expression testing and pathway analysis with the results obtained informing future experiments and validation studies. In this workflow article, we analyse RNA-sequencing data from the mouse mammary gland, demonstrating use of the popular edgeR package to import, organise, filter and normalise the data, followed by the limma package with its voom method, linear modelling and empirical Bayes moderation to assess differential expression and perform gene set testing. This pipeline is further enhanced by the Glimma package which enables interactive exploration of the results so that in idual s les and genes can be examined by the user. The complete analysis offered by these three packages highlights the ease with which researchers can turn the raw counts from an RNA-sequencing experiment into biological insights using Bioconductor.
Publisher: Springer Science and Business Media LLC
Date: 2010
Publisher: The Company of Biologists
Date: 2015
DOI: 10.1242/DEV.122077
Abstract: Epigenetic mechanisms involved in the establishment of lung epithelial cell lineage identities during development are largely unknown. Here, we explored the role of the histone methyltransferase Ezh2 during lung lineage determination. Loss of Ezh2 in the lung epithelium leads to defective lung formation and perinatal mortality. We show that Ezh2 is crucial for airway lineage specification and alveolarization. Using optical projection tomography imaging, we found that branching morphogenesis is affected in Ezh2 conditional knockout mice and the remaining bronchioles are abnormal, lacking terminally differentiated secretory club cells. Remarkably, RNA-seq analysis revealed the upregulation of basal genes in Ezh2-deficient epithelium. Three-dimensional imaging for keratin 5 further showed the unexpected presence of a layer of basal cells from the proximal airways to the distal bronchioles in E16.5 embryos. ChIP-seq analysis indicated the presence of Ezh2-mediated repressive marks on the genomic loci of some but not all basal genes, suggesting an indirect mechanism of action of Ezh2. We found that loss of Ezh2 de-represses insulin-like growth factor 1 (Igf1) expression and that modulation of IGF1 signaling ex vivo in wild-type lungs could induce basal cell differentiation. Altogether, our work reveals an unexpected role for Ezh2 in controlling basal cell fate determination in the embryonic lung endoderm, mediated in part by repression of Igf1 expression.
Publisher: Springer Science and Business Media LLC
Date: 06-02-2008
Publisher: Elsevier BV
Date: 2023
Publisher: Oxford University Press (OUP)
Date: 15-02-2017
DOI: 10.1093/BIOINFORMATICS/BTX094
Abstract: Summary graphics for RNA-sequencing and microarray gene expression analyses may contain upwards of tens of thousands of points. Details about certain genes or s les of interest are easily obscured in such dense summary displays. Incorporating interactivity into summary plots would enable additional information to be displayed on demand and facilitate intuitive data exploration. The open-source Glimma package creates interactive graphics for exploring gene expression analysis with a few simple R commands. It extends popular plots found in the limma package, such as multi-dimensional scaling plots and mean-difference plots, to allow in idual data points to be queried and additional annotation information to be displayed upon hovering or selecting particular points. It also offers links between plots so that more information can be revealed on demand. Glimma is widely applicable, supporting data analyses from a number of well-established Bioconductor workflows (limma, edgeR and DESeq2) and uses D3/JavaScript to produce HTML pages with interactive displays that enable more effective data exploration by end-users. Results from Glimma can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility. The Glimma R package is available from ackages/Glimma/.
Publisher: Cold Spring Harbor Laboratory
Date: 21-10-2021
DOI: 10.1101/2021.10.21.465360
Abstract: 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 embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1’s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.
Publisher: Oxford University Press (OUP)
Date: 09-2020
Abstract: 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 informative, and through exploratory data analysis of read coverage that intron signal is representative of both pre-mRNAs and intron retention. We demonstrate how intron reads can be utilized in differential expression analysis using our index method where a unique set of differentially expressed genes can be detected using intron counts. In exploring read coverage, we also developed the superintronic software that quickly and robustly calculates user-defined summary statistics for exonic and intronic regions. Across multiple datasets, superintronic enabled us to identify several genes with distinctly retained introns that had similar coverage levels to that of neighbouring exons. The work and ideas presented in this paper is the first of its kind to consider multiple biological sources for intron reads through exploratory data analysis, minimizing bias in discovery and interpretation of results. Our findings open up possibilities for further methods development for intron reads and RNA-seq data in general.
Publisher: Elsevier BV
Date: 12-2016
Publisher: Elsevier BV
Date: 12-2014
Publisher: Springer Science and Business Media LLC
Date: 31-07-2008
Abstract: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or lification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ . A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.
Publisher: Springer Science and Business Media LLC
Date: 23-05-2014
Publisher: Cold Spring Harbor Laboratory
Date: 08-2021
DOI: 10.1101/2021.07.30.454464
Abstract: 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 inter-activity and reproducibility using high-level visualisation frame-works for R and JavaScript. Glimma 2.0 plots are now readily embeddable in R Markdown, thus allowing users to create reproducible reports containing interactive graphics. The rev ed multidimensional scaling plot features dashboard-style controls allowing the user to dynamically change the colour, shape and size of s le points according to different experimental conditions. Interactivity was enhanced in the MA-style plot for comparing differences to average expression, which now supports selecting multiple genes, export options to PNG, SVG or CSV formats and includes a new volcano plot function. Feature-rich and user-friendly, Glimma makes exploring data for gene expression analysis more accessible and intuitive and is available on Bioconductor and GitHub .
Publisher: Cold Spring Harbor Laboratory
Date: 26-03-2022
DOI: 10.1101/2022.03.25.485728
Abstract: Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals including mice, rats, rabbits and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been “the gold standard” for antibody gene sequencing but relies on the availability of species-specific degenerate primer sets for lification of light and heavy antibody genes, in addition to lengthy and expensive cDNA preparation. Here we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop NAb-seq: a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light and heavy chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics and therapeutics.
Publisher: Rockefeller University Press
Date: 21-09-2022
Abstract: MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B–related metabolite antigens from erse 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 host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-06-2015
Abstract: Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a protein that plays an important role in maintaining gene silencing in many biological circumstances, including facioscapulohumeral muscular dystrophy however, how it brings about gene silencing is unknown. Understanding the molecular mechanism by which Smchd1 contributes to stable transcriptional silencing is critical to appreciate how it functions in normal biology and when it is mutated in facioscapulohumeral muscular dystrophy. This study reveals, for the first time to our knowledge, where Smchd1 binds genome-wide, its hitherto unappreciated functional interaction with chromatin organizer CCCTC-binding factor in gene regulation, and which part of the protein is required for chromatin binding. These data lead to a new model of Smchd1 function, where it directly binds DNA to mediate 3D chromatin architecture.
Publisher: Oxford University Press (OUP)
Date: 25-08-2007
DOI: 10.1093/BIOINFORMATICS/BTM412
Abstract: Motivation: Microarray data must be background corrected to remove the effects of non-specific binding or spatial heterogeneity across the array, but this practice typically causes other problems such as negative corrected intensities and high variability of low intensity log-ratios. Different estimators of background, and various model-based processing methods, are compared in this study in search of the best option for differential expression analyses of small microarray experiments. Results: Using data where some independent truth in gene expression is known, eight different background correction alternatives are compared, in terms of precision and bias of the resulting gene expression measures, and in terms of their ability to detect differentially expressed genes as judged by two popular algorithms, SAM and limma eBayes. A new background processing method (normexp) is introduced which is based on a convolution model. The model-based correction methods are shown to be markedly superior to the usual practice of subtracting local background estimates. Methods which stabilize the variances of the log-ratios along the intensity range perform the best. The normexp+offset method is found to give the lowest false discovery rate overall, followed by morph and vsn. Like vsn, normexp is applicable to most types of two-colour microarray data. Availability: The background correction methods compared in this article are available in the R package limma (Smyth, 2005) from www.bioconductor.org. Contact: smyth@wehi.edu.au Supplementary information: Supplementary data are available from bioinf.wehi.edu.au/resources/webReferences.html.
Publisher: American Society of Hematology
Date: 15-06-2023
DOI: 10.1182/BLOODADVANCES.2022008221
Abstract: 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 are limited. Combining venetoclax with immunotherapies is an attractive approach however, a detailed understanding of how venetoclax treatment impacts normal immune cells in patients is lacking. In this study, we performed deep profiling of peripheral blood (PB) cells from patients with CLL and AML before and after short-term treatment with venetoclax using mass cytometry (cytometry by time of flight) and found no impact on the concentrations of key T-cell subsets or their expression of checkpoint molecules. We also analyzed PB from patients with breast cancer receiving venetoclax long-term using a single-cell multiomics approach (cellular indexing of transcriptomes and epitopes by sequencing) and functional assays. We found significant depletion of B-cell populations with low expression of MCL-1 relative to other immune cells, attended by extensive transcriptomic changes. By contrast, there was less impact on circulating T cells and natural killer (NK) cells, with no changes in their subset composition, transcriptome, or function following venetoclax treatment. Our data indicate that venetoclax has minimal impact on circulating T or NK cells, supporting the rationale of combining this BH3 mimetic drug with cancer immunotherapies for more durable antitumor responses.
Publisher: F1000 Research Ltd
Date: 04-12-2013
DOI: 10.12688/F1000RESEARCH.2-264.V1
Abstract: The IDAT file format is used to store BeadArray data from the myriad of genomewide profiling platforms on offer from Illumina Inc. This proprietary format is output directly from the scanner and stores summary intensities for each probe-type on an array in a compact manner. A lack of open source tools to process IDAT files has h ered their uptake by the research community beyond the standard step of using the vendor’s software to extract the data they contain in a human readable text format. To fill this void, we have developed the illuminaio package that parses IDAT files from any BeadArray platform, including the decryption of files from Illumina’s gene expression arrays. illuminaio provides the first open-source package for this task, and will promote wider uptake of the IDAT format as a standard for sharing Illumina BeadArray data in public databases, in the same way that the CEL file serves as the standard for the Affymetrix platform.
Publisher: Cold Spring Harbor Laboratory
Date: 25-09-2023
Publisher: Oxford University Press (OUP)
Date: 22-02-2019
DOI: 10.1093/NAR/GKZ107
Publisher: Cold Spring Harbor Laboratory
Date: 17-10-2018
DOI: 10.1101/445924
Abstract: Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of approximately ten, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterise, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new imprinting control regions.
Publisher: Springer Science and Business Media LLC
Date: 19-10-2019
DOI: 10.1007/S12035-019-01782-8
Abstract: 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 processes and elicit a erse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.
Publisher: Cold Spring Harbor Laboratory
Date: 13-05-2021
DOI: 10.1101/2021.05.12.443934
Abstract: 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 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against another epigenetic regulator complex, PRC2, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1’s role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.
Publisher: Proceedings of the National Academy of Sciences
Date: 18-07-2012
Abstract: Overexpression of the prosurvival protein BCL-2 is common in breast cancer. Here we have explored its role as a potential therapeutic target in this disease. BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the proapoptotic BH3-only ligand BIM were found to be coexpressed at relatively high levels in a substantial proportion of heterogeneous breast tumors, including clinically aggressive basal-like cancers. To determine whether the BH3 mimetic ABT-737 that neutralizes BCL-2, BCL-XL, and BCL-W had potential efficacy in targeting BCL-2–expressing basal-like triple-negative tumors, we generated a panel of primary breast tumor xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel, or a combination. Tumor response and overall survival were significantly improved by combination therapy, but only for tumor xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumor cells to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2. Notably, BH3 mimetics also appeared effective in BCL-2–expressing xenograft lines that harbored p53 mutations. Our findings provide in vivo evidence that BH3 mimetics can be used to sensitize primary breast tumors to chemotherapy and further suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer.
Publisher: Oxford University Press (OUP)
Date: 06-08-2009
DOI: 10.1093/BIOINFORMATICS/BTP470
Abstract: Summary: Illumina produces a number of microarray-based technologies for human genotyping. An Infinium BeadChip is a two-color platform that types between 105 and 106 single nucleotide polymorphisms (SNPs) per s le. Despite being widely used, there is a shortage of open source software to process the raw intensities from this platform into genotype calls. To this end, we have developed the R/Bioconductor package crlmm for analyzing BeadChip data. After careful preprocessing, our software applies the CRLMM algorithm to produce genotype calls, confidence scores and other quality metrics at both the SNP and s le levels. We provide access to the raw summary-level intensity data, allowing users to develop their own methods for genotype calling or copy number analysis if they wish. Availability and Implementation: The crlmm Bioconductor package is available from www.bioconductor.org. Data packages and documentation are available from oftware.html. Contact: mritchie@wehi.edu.au rafa@jhu.edu
Publisher: Springer Science and Business Media LLC
Date: 19-10-2020
DOI: 10.1038/S41590-020-0799-X
Abstract: A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.YJMCC.2007.11.020
Abstract: Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying mechanisms linking these conditions is yet to be determined. Using a 'gene dose' Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, >95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8+/-13.2 mmHg vs. WT, 146.4+/-9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic beta-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional 'rescue'.
Publisher: American Society of Hematology
Date: 17-11-2022
Abstract: Venetoclax (VEN) inhibits the prosurvival 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 subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and lification of the MCL1 gene but is heterogeneous across and within in idual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.
Publisher: F1000 Research Ltd
Date: 21-10-2014
DOI: 10.12688/F1000RESEARCH.3928.2
Abstract: Pooled library sequencing screens that perturb gene function in a high-throughput manner are becoming increasingly popular in functional genomics research. Irrespective of the mechanism by which loss of function is achieved, via either RNA interference using short hairpin RNAs (shRNAs) or genetic mutation using single guide RNAs (sgRNAs) with the CRISPR-Cas9 system, there is a need to establish optimal analysis tools to handle such data. Our open-source processing pipeline in edgeR provides a complete analysis solution for screen data, that begins with the raw sequence reads and ends with a ranked list of candidate genes for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (s les in the columns, genes in the rows) with options for allowing mismatches and small shifts in sequence position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or generalized linear modeling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from www.bioconductor.org.
Publisher: Cold Spring Harbor Laboratory
Date: 04-04-2023
DOI: 10.1101/2023.04.02.535231
Abstract: 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 sequence reads to such transcripts. The quantification uncertainty associated with transcript-level estimated counts is intractable to measure analytically but represents an extra source of variation that seriously compromises differential transcript expression (DTE) analyses if standard statistical methods developed for gene-level analyses are used. Bootstrap counts, as provided by popular RNA-seq quantification tools, allow one to estimate the quantification uncertainty and account for such an effect in DTE analyses. We present catchSalmon and catchKallisto , two functions included in the R/Bioconductor package edgeR , that estimate the transcript-level quantification uncertainty, here termed mapping ambiguity overdispersion, using bootstrap counts. We discuss how the mapping ambiguity overdispersion can be effectively removed from the data in transcript-level analyses via count scaling, an approach that reduces the size of the estimated counts obtained from quantification tools to effective count sizes that reflect their true precision. The presented count scaling approach allows users to perform efficient DTE analyses within the efficient edgeR framework. A comprehensive simulation study and a DTE analysis of human lung adenocarcinoma cell lines are presented to illustrate the benefits of accounting for the mapping ambiguity overdispersion in transcript-level RNA-seq data analyses.
Publisher: F1000 Research Ltd
Date: 24-04-2014
DOI: 10.12688/F1000RESEARCH.3928.1
Abstract: Pooled short hairpin RNA sequencing (shRNA-seq) screens are becoming increasingly popular in functional genomics research, and there is a need to establish optimal analysis tools to handle such data. Our open-source shRNA processing pipeline in edgeR provides a complete analysis solution for shRNA-seq screen data, that begins with the raw sequence reads and ends with a ranked lists of candidate shRNAs for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (s les in the columns, hairpins in the rows) with options for allowing mismatches and small shifts in hairpin position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or a generalized linear modelling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from www.bioconductor.org .
Publisher: Cold Spring Harbor Laboratory
Date: 03-05-2023
DOI: 10.1101/2023.05.02.539165
Abstract: 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 PCR can also introduce artefacts which should be flagged in data pre-processing. Here we introduce Restrander , a lightning-fast and highly accurate tool for restranding and quality checking long-read cDNA sequencing data. Thanks to its C++ implementation, Restrander was faster than Oxford Nanopore Technologies’ existing tool Pychopper , and correctly restranded more reads due to its strategy of searching for polyA/T tails in addition to primer sequences from the reverse transcription and template-switch steps. We found that restranding improved the process of visualising and exploring data, and increased the number of novel isoforms discovered by bambu , particularly in regions where sense and antisense transcripts co-occur. The artefact detection implemented in Restrander quantifies reads which do not have the correct 5’ and 3’ ends, a feature which is useful in quality control for library preparation. Restrander is pre-configured for all major cDNA protocols, and can be customised with user-defined primers. Restrander is available at akob-schuster/restrander
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.CCR.2013.06.002
Abstract: The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer.
Publisher: Japanese Society of Hypertension
Date: 2008
Abstract: The hypertrophic heart rat (HHR) was derived from the spontaneously hypertensive rat of the Okamoto strain and develops cardiac hypertrophy in the absence of hypertension. The genetic basis of this hypertrophy is unknown. Therefore, we compared gene expression profiles in the left ventricular myocardium of young (8-10 weeks of age) and old (38-50 weeks) HHR with rats from an age-matched control strain, the normal heart rat (NHR). cDNA microarrays (National Institute of Aging [NIA], 15,247 clones) were used to evaluate gene expression in cardiac-derived Cy3 and Cy5 labeled cDNA. M values (log2[Cy5/Cy3]) were obtained and significant differential expression was identified using an empirical Bayesian approach with specific results verified using real-time PCR. Compared with NHR, HHR cardiac weight index (heart weight/ body weight) was significantly elevated at both ages (young: 5.5 +/- 0.5 vs. 3.9 +/- 0.2 old: 4.2 +/- 0.3 vs. 3.4 +/- 0.2 mg/g p < 0.05) with no difference in body weight or in tail-cuff blood pressure detected between the strains at either age. Differential expression was observed in 65 and 390 clones in young and old HHR, respectively, with more genes exhibiting down-regulation than up-regulation in both instances (young: down 44 vs. up 21 old: down 292 vs. up 98). Our data suggest a role for the Ras/mitogen-activated protein kinase (MAPK) signaling pathway and the tumor necrosis factor (TNF) receptor-mediated activation of nuclear factor-kappaB (NF-kappaB) in the etiology of cardiac enlargement in the HHR. These findings support the candidature of previously identified cardiotrophic agents in contributing to the cardiac enlargement in the normotensive HHR, and also identify novel genetic factors which may be involved in the genesis of primary cardiac hypertrophy.
Publisher: Springer Science and Business Media LLC
Date: 29-03-2022
DOI: 10.1038/S41467-022-29333-1
Abstract: 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 compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.
Publisher: F1000 Research Ltd
Date: 29-05-2019
DOI: 10.12688/F1000RESEARCH.19410.1
Abstract: Motivation: The Bioconductor project, a large collection of open source software for the comprehension of large-scale biological data, continues to grow with new packages added each week, motivating the development of software tools focused on exposing package metadata to developers and users. The resulting BiocPkgTools package facilitates access to extensive metadata in computable form covering the Bioconductor package ecosystem, facilitating downstream applications such as custom reporting, data and text mining of Bioconductor package text descriptions, graph analytics over package dependencies, and custom search approaches. Results: The BiocPkgTools package has been incorporated into the Bioconductor project, installs using standard procedures, and runs on any system supporting R. It provides functions to load detailed package metadata, longitudinal package download statistics, package dependencies, and Bioconductor build reports, all in "tidy data" form. BiocPkgTools can convert from tidy data structures to graph structures, enabling graph-based analytics and visualization. An end-user-friendly graphical package explorer aids in task-centric package discovery. Full documentation and ex le use cases are included. Availability: The BiocPkgTools software and complete documentation are available from Bioconductor ( ackages/BiocPkgTools ).
Publisher: Public Library of Science (PLoS)
Date: 25-10-2021
DOI: 10.1371/JOURNAL.PCBI.1009524
Abstract: A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between s les from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the s le-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across in idual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at ackages/NanoMethViz .
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2020
DOI: 10.1101/2020.01.20.913376
Abstract: 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 the epigenetic repressor Smchd1 as a novel maternal effect gene that regulates imprinted expression of 16 genes. Most Smchd1-sensitive genes only show loss of imprinting post-implantation, indicating maternal Smchd1’s long-lived epigenetic effect. Sm-chd1-sensitive genes include both those controlled by germline polycomb marks and germline DNA methylation imprints however, Smchd1 differs to other maternal effect genes that regulate the latter group, as Smchd1 does not affect germline DNA methylation imprints. Instead, Smchd1-sensitive genes are united by their reliance on polycomb-mediated histone methylation marks as germline or secondary imprints. We propose that Smchd1 translates these imprints to establish a heritable chromatin state required for imprinted expression later in development, revealing a new mechanism for maternal effect genes.
Publisher: Public Library of Science (PLoS)
Date: 16-07-2010
Publisher: Informa UK Limited
Date: 13-08-2022
DOI: 10.1080/19420862.2022.2106621
Abstract: Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing, but this method relies on the availability of species-specific degenerate primer sets for lification of light and heavy antibody genes and it requires lengthy and expensive cDNA preparation. Here, we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop Nanopore Antibody sequencing (NAb-seq): a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light- and heavy-chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics, and therapeutics.
Publisher: Oxford University Press (OUP)
Date: 23-06-2009
DOI: 10.1093/BIOINFORMATICS/BTP383
Abstract: Motivation: Primary data analysis methods are of critical importance in second generation DNA sequencing. Improved methods have the potential to increase yield and reduce the error rates. Openly documented analysis tools enable the user to understand the primary data, this is important for the optimization and validity of their scientific work. Results: In this article, we describe Swift, a new tool for performing primary data analysis on the Illumina Solexa Sequencing Platform. Swift is the first tool, outside of the vendors own software, which completes the full analysis process, from raw images through to base calls. As such it provides an alternative to, and independent validation of, the vendor supplied tool. Our results show that Swift is able to increase yield by 13.8%, at comparable error rate. Availability and Implementation: Swift is implemented in C++and supported under Linux. It is supplied under an open source license (LGPL3), allowing researchers to build upon the platform. Swift is available from swiftng.sourceforge.net. Contact: new@sgenomics.org nava.whiteford@nanoporetech.com Supplementary information: Supplementary data are available at Bioinformatics online.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2019
DOI: 10.1038/S41592-019-0425-8
Abstract: Single cell RNA-sequencing (scRNA-seq) technology has undergone rapid development in recent years, leading to an explosion in the number of tailored data analysis methods. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically compare the performance of the many methods available. Here, we generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create 'pseudo cells' from up to five distinct cancer cell lines. In total, 14 datasets were generated using both droplet and plate-based scRNA-seq protocols. We compared 3,913 combinations of data analysis methods for tasks ranging from normalization and imputation to clustering, trajectory analysis and data integration. Evaluation revealed pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps.
Publisher: Springer Science and Business Media LLC
Date: 19-05-2006
Abstract: Assessment of array quality is an essential step in the analysis of data from microarray experiments. Once detected, less reliable arrays are typically excluded or "filtered" from further analysis to avoid misleading results. In this article, a graduated approach to array quality is considered based on empirical reproducibility of the gene expression measures from replicate arrays. Weights are assigned to each microarray by fitting a heteroscedastic linear model with shared array variance terms. A novel gene-by-gene update algorithm is used to efficiently estimate the array variances. The inverse variances are used as weights in the linear model analysis to identify differentially expressed genes. The method successfully assigns lower weights to less reproducible arrays from different experiments. Down-weighting the observations from suspect arrays increases the power to detect differential expression. In smaller experiments, this approach outperforms the usual method of filtering the data. The method is available in the limma software package which is implemented in the R software environment. This method complements existing normalisation and spot quality procedures, and allows poorer quality arrays, which would otherwise be discarded, to be included in an analysis. It is applicable to microarray data from experiments with some level of replication.
Publisher: Springer Science and Business Media LLC
Date: 18-07-2022
DOI: 10.1186/S13072-022-00458-3
Abstract: 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 embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1’s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.
Publisher: Oxford University Press (OUP)
Date: 22-06-2007
DOI: 10.1093/BIOINFORMATICS/BTM311
Abstract: Summary: The R/Bioconductor package beadarray allows raw data from Illumina experiments to be read and stored in convenient R classes. Users are free to choose between various methods of image processing, background correction and normalization in their analysis rather than using the defaults in Illumina's proprietary software. The package also allows quality assessment to be carried out on the raw data. The data can then be summarized and stored in a format which can be used by other R/Bioconductor packages to perform downstream analyses. Summarized data processed by Illumina's BeadStudio software can also be read and analysed in the same manner. Availability: The beadarray package is available from the Bioconductor web page at www.bioconductor.org. A user's guide and ex le data sets are provided with the package. Contact: md392@cam.ac.uk
Publisher: American Association for Cancer Research (AACR)
Date: 08-2017
DOI: 10.1158/1535-7163.MCT-17-0174
Abstract: Lung squamous cell carcinoma (SqCC) is a molecularly complex and genomically unstable disease. No targeted therapy is currently approved for lung SqCC, although potential oncogenic drivers of SqCC have been identified, including lification of the fibroblast growth factor receptor 1 (FGFR1). Reports from a recently completed clinical trial indicate low response rates in patients treated with FGFR tyrosine kinase inhibitors, suggesting inadequacy of FGFR1 lification as a biomarker of response, or the need for combination treatment. We aimed to develop accurate models of lung SqCC and determine improved targeted therapies for these tumors. We show that detection of FGFR1 mRNA by RNA in situ hybridization is a better predictor of response to FGFR inhibition than FGFR1 gene lification using clinically relevant patient-derived xenograft (PDX) models of lung SqCC. FGFR1-overexpressing tumors were observed in all histologic subtypes of non–small cell lung cancers (NSCLC) as assessed on a tissue microarray, indicating a broader range of tumors that may respond to FGFR inhibitors. In FGFR1-overexpressing PDX tumors, we observed increased differentiation and reduced proliferation following FGFR inhibition. Combination therapy with cisplatin was able to increase tumor cell death, and dramatically prolonged animal survival compared to single-agent treatment. Our data suggest that FGFR tyrosine kinase inhibitors can benefit NSCLC patients with FGFR1-overexpressing tumors and provides a rationale for clinical trials combining cisplatin with FGFR inhibitors. Mol Cancer Ther 16(8) 1610–22. ©2017 AACR.
Publisher: Springer Science and Business Media LLC
Date: 15-05-2020
DOI: 10.1038/S41467-020-16214-8
Abstract: 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 archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells.
Publisher: Springer Science and Business Media LLC
Date: 02-10-2023
Publisher: Springer Science and Business Media LLC
Date: 13-07-2022
DOI: 10.1038/S41418-022-01037-5
Abstract: 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 in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1 , Myof , Gas7 and Atoh8 . Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.
Publisher: Cold Spring Harbor Laboratory
Date: 03-10-2018
DOI: 10.1101/433102
Abstract: Single cell RNA sequencing (scRNA-seq) technology has undergone rapid development in recent years, bringing with new challenges in data processing and analysis. This has led to an explosion of tailored analysis methods for scRNA-seq data to address various biological questions. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically evaluate the performance of the many methods available. Here, we designed and carried out a realistic benchmark experiment that included mixtures of single cells or ‘pseudo cells’ created by s ling admixtures of cells or RNA from up to 5 distinct cancer cell lines. Altogether we generated 14 datasets using droplet and plate-based scRNA-seq protocols, compared multiple data analysis methods in combination for tasks ranging from normalization and imputation, to clustering, trajectory analysis and data integration. Evaluation across 3,913 analyses (methods × benchmark dataset combinations) revealed pipelines suited to different types of data for different tasks. Our dataset and analysis present a comprehensive comparison framework for benchmarking most common scRNA-seq analysis tasks.
Publisher: Cold Spring Harbor Laboratory
Date: 29-08-2018
DOI: 10.1101/403113
Abstract: Conventional single cell RNA-seq methods are destructive, such that a given cell cannot also then be tested for fate and function, without a time machine. Here, we develop a clonal method SIS-seq , whereby single cells are allowed to ide, and progeny cells are assayed separately in SIS ter conditions some for fate, others by RNA- seq . By cross-correlating progenitor gene expression with mature cell fate within a clone, and doing this for many clones, we can identify the earliest gene expression signatures of dendritic cell subset development. SIS-seq could be used to study other populations harboring clonal heterogeneity, including stem, reprogrammed and cancer cells to reveal the transcriptional origins of fate decisions.
Publisher: Radiation Research Society
Date: 27-09-2017
DOI: 10.1667/RR14794.1
Publisher: Oxford University Press (OUP)
Date: 08-12-2008
Publisher: Public Library of Science (PLoS)
Date: 10-08-2018
Publisher: Oxford University Press (OUP)
Date: 21-02-2012
DOI: 10.1093/BIOINFORMATICS/BTS089
Abstract: Motivation: In the past decade, a number of technologies to quantify allele-specific expression (ASE) in a genome-wide manner have become available to researchers. We investigate the application of single-nucleotide polymorphism (SNP) microarrays to this task, exploring data obtained from both cell lines and primary tissue for which both RNA and DNA profiles are available. Results: We analyze data from two experiments that make use of high-density Illumina Infinium II genotyping arrays to measure ASE. We first preprocess each data set, which involves removal of outlier s les, careful normalization and a two-step filtering procedure to remove SNPs that show no evidence of expression in the s les being analyzed and calls that are clear genotyping errors. We then compare three different tests for detecting ASE, one of which has been previously published and two novel approaches. These tests vary at the level at which they operate (per SNP per in idual or per SNP) and in the input data they require. Using SNPs from imprinted genes as true positives for ASE, we observe varying sensitivity for the different testing procedures that improves with increasing s le size. Methods that rely on RNA signal alone were found to perform best across a range of metrics. The top ranked SNPs recovered by all methods appear to be reasonable candidates for ASE. Availability and implementation: Analysis was carried out in R (www.R-project.org/) using existing functions. Contact: mritchie@wehi.edu.au. Supplementary information: Supplementary data are available at Bioinformatics online.
Publisher: Cold Spring Harbor Laboratory
Date: 08-08-2019
DOI: 10.1101/729400
Abstract: ILC3-mediated IL-22 cytokine production is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we show that group 3 ILC (ILC3) constitutive function is not constant across the day but instead oscilliates between active and resting phases. Coordinate responsiveness of ILC3 in the intestine depended on food-induced expression of the neuronal hormone vasoactive intestinal peptide (VIP). Intestinal ILC3 expressed high levels of the G protein-coupled receptor, VIPR2, and activation via enteric neuronal VIP markedly enhanced IL-22 production and conferred gut protection. Conversely, deficiency of VIPR2 signalling led to impaired production of IL-22 by ILC3 and increased susceptibility to inflammatory gut disease. As such, intrinsic cellular rhythms synergise with the cyclic patterns of food intake to drive IL-22 thereby syncronizing intestinal epithelial protection via the ILC3 VIP-VIPR2 pathway.
Publisher: Springer Science and Business Media LLC
Date: 04-06-2008
Abstract: Variance-stabilizing techniques have been used for some time in the analysis of gene expression microarray data. A new adaptation, the variance-stabilizing transformation (VST), has recently been developed to take advantage of the unique features of Illumina BeadArrays. VST has been shown to perform well in comparison with the widely-used approach of taking a log 2 transformation, but has not been validated on a spike-in experiment. We apply VST to the data from a recently published spike-in experiment and compare it both to a regular log 2 analysis and a recently recommended analysis that can be applied if all raw data are available. VST provides more power to detect differentially expressed genes than a log 2 transformation. However, the gain in power is roughly the same as utilizing the raw data from an experiment and weighting observations accordingly. VST is still advantageous when large changes in expression are anticipated, while a weighted log 2 approach performs better for smaller changes. VST can be recommended for summarized Illumina data regardless of which Illumina pre-processing options have been used. However, using the raw data is still encouraged whenever possible.
Publisher: Informa UK Limited
Date: 2006
DOI: 10.1080/00016480600702118
Abstract: Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. To gain understanding of the gene pathways involved in the development of the normal cochlea. Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.
Publisher: SAGE Publications
Date: 19-01-2009
Abstract: Illumina’s GoldenGate technology is a two-channel microarray platform that allows for the simultaneous interrogation of about 1 500 locations in the genome. GoldenGate has proved a flexible platform not only in the choice of those 1 500 locations, but also in the choice of the property being measured at them. It retains the desirable properties of Illumina’s BeadArrays in that the probes (in this case ‘beads’) are randomly arranged across the microarray, there are multiple instances of each probe and many s les can be processed simultaneously. As for other Illumina technologies, however, these properties are not exploited as they might be. Here we review the various common adaptations of the GoldenGate platform, review the analysis methods that are associated with each adaptation and then, with the aid of a number of ex le data sets we illustrate some of the improvements that can be made over the default analysis.
Publisher: Springer Science and Business Media LLC
Date: 23-12-2020
DOI: 10.1038/S41590-019-0567-Y
Abstract: Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.
Publisher: Cold Spring Harbor Laboratory
Date: 10-08-2020
DOI: 10.1101/2020.08.10.243543
Abstract: 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 s ling ( FLT-seq ), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES , we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on in idual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.
Publisher: The Company of Biologists
Date: 07-2021
DOI: 10.1242/DEV.199543
Abstract: Development of a branching tree in the embryonic lung is crucial 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 in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.
Publisher: Public Library of Science (PLoS)
Date: 12-2011
Publisher: F1000 Research Ltd
Date: 14-11-2017
DOI: 10.12688/F1000RESEARCH.12544.1
Abstract: Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from in idual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.
Publisher: American Society of Hematology
Date: 05-08-2010
DOI: 10.1182/BLOOD-2009-12-260760
Abstract: Polycomb group (PcG) proteins are transcriptional repressors with a central role in the establishment and maintenance of gene expression patterns during development. We have investigated the role of polycomb repressive complexes (PRCs) in hematopoietic stem cells (HSCs) and progenitor populations. We show that mice with loss of function mutations in PRC2 components display enhanced HSC rogenitor population activity, whereas mutations that disrupt PRC1 or pleiohomeotic repressive complex are associated with HSC rogenitor cell defects. Because the hierarchical model of PRC action would predict synergistic effects of PRC1 and PRC2 mutation, these opposing effects suggest this model does not hold true in HSC rogenitor cells. To investigate the molecular targets of each complex in HSC rogenitor cells, we measured genome-wide expression changes associated with PRC deficiency, and identified transcriptional networks that are differentially regulated by PRC1 and PRC2. These studies provide new insights into the mechanistic interplay between distinct PRCs and have important implications for approaching PcG proteins as therapeutic targets.
Publisher: American Society of Hematology
Date: 04-2020
DOI: 10.1182/BLOODADVANCES.2019001323
Abstract: 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 contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.
Publisher: Elsevier BV
Date: 04-2022
Publisher: Elsevier BV
Date: 03-2016
Publisher: Springer Science and Business Media LLC
Date: 13-02-2017
DOI: 10.1038/NCB3471
Abstract: Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5
Publisher: SAGE Publications
Date: 18-01-2017
Abstract: Terrestrial cosmogenic nuclide dating has been widely used to estimate the surface exposure age of bedrock and boulder surfaces associated with deglaciation and Holocene glacier variations, but the effect of inherited age has been rarely directly addressed. In this study, small clasts, embedded in flute surfaces on two cirque glacier forelands in Jotunheimen, southern Norway and deposited within the last ~60 years, were used to test whether such clasts have the modern surface exposure age expected in the absence of inheritance. Two different approaches were taken involving dating of (1) a single clast of cobble size from the proglacial area of Austanbotnbreen, and (2) 75 clasts mostly of pebble size from the proglacial area of Storbreen crushed and treated as a single s le. 10 Be surface exposure ages were 99 ± 98 and 368 ± 90 years, respectively, with 95% confidence (±2σ). It is concluded that (1) these small glaciers have eroded and deposited rock fragments with a cosmogenic zero or near-zero concentration, (2) the likelihood of inherited cosmogenic nuclide concentrations in similar rock fragments deposited by larger warm-based glaciers and ice sheets should be small, and (3) combining a large number of small rock particles into one s le rather than using single large clasts of boulder size may provide a viable alternative to the commonly perceived need for five or more independent estimates of exposure age per site.
Publisher: Oxford University Press (OUP)
Date: 25-02-2023
DOI: 10.1093/NAR/GKAD091
Abstract: 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 methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a erse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.
Publisher: Cold Spring Harbor Laboratory
Date: 21-08-2023
DOI: 10.1101/2023.08.21.554084
Abstract: 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, the existing tools which perform these functions are often task-specific, such as only demultiplexing barcodes for a dedicated type of experiment or are not tolerant to noise in the sequencing data. To overcome these limitations, we developed Flexiplex, a versatile and fast sequence searching and demultiplexing tool for omics data, which is based on the Levenshtein distance and thus allows imperfect matches. We demonstrate Flexiplex’s application on three use cases, identifying cell line specific sequences in Illumina short read single cell data, and discovering and demultiplexing cellular barcodes from noisy long-read single cell RNA-seq data. We show that Flexiplex achieves an excellent balance of accuracy and computational efficiency compared to leading task-specific tools. Flexiplex is available at davidsongroup.github.io/flexiplex/ .
Publisher: Springer Science and Business Media LLC
Date: 19-04-2010
Abstract: Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331 . None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2 , a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more in iduals in 39 candidate genes (18%). Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.
Publisher: Oxford University Press (OUP)
Date: 30-09-2016
DOI: 10.1093/BIOINFORMATICS/BTW623
Abstract: Gene set enrichment (GSE) analysis allows researchers to efficiently extract biological insight from long lists of differentially expressed genes by interrogating them at a systems level. In recent years, there has been a proliferation of GSE analysis methods and hence it has become increasingly difficult for researchers to select an optimal GSE tool based on their particular dataset. Moreover, the majority of GSE analysis methods do not allow researchers to simultaneously compare gene set level results between multiple experimental conditions. The ensemble of genes set enrichment analyses (EGSEA) is a method developed for RNA-sequencing data that combines results from twelve algorithms and calculates collective gene set scores to improve the biological relevance of the highest ranked gene sets. EGSEA’s gene set database contains around 25 000 gene sets from sixteen collections. It has multiple visualization capabilities that allow researchers to view gene sets at various levels of granularity. EGSEA has been tested on simulated data and on a number of human and mouse datasets and, based on biologists’ feedback, consistently outperforms the in idual tools that have been combined. Our evaluation demonstrates the superiority of the ensemble approach for GSE analysis, and its utility to effectively and efficiently extrapolate biological functions and potential involvement in disease processes from lists of differentially regulated genes. EGSEA is available as an R package at ackages/EGSEA/. The gene sets collections are available in the R package EGSEAdata from ackages/EGSEAdata/. Supplementary data are available at Bioinformatics online.
Location: Australia
Start Date: 09-2021
End Date: 09-2024
Amount: $517,138.00
Funder: Australian Research Council
View Funded ActivityStart Date: 02-2020
End Date: 12-2023
Amount: $650,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 09-2018
End Date: 08-2021
Amount: $615,502.00
Funder: Australian Research Council
View Funded Activity