Pre-Event Focus Day Workshop

03 November 2020 | 14:00 – 17:00 BST | 15:00 - 18:00 CET

Join our Diamond Sponsor Takara Bio for a complimentary pre-event focus day workshop

Investigating Human Health And Disease At Single-Cell Resolution

Workshop Overview
Single-cell genomics is revolutionizing our understanding of biology by providing decisive insights into the complexity of cells and tissues in model organisms and in humans.
This workshop is bringing together a panel of worldwide experts in single genomics to discuss how human health and disease can be resolved at the single-cell level. Participants will hear how advances in single-cell genomics are leading to a better understanding of complex biological processes at play in various diseases and may pave the way for innovative therapeutic intervention. The many promises and current challenges in bringing single-cell omics in the clinic will also be discussed.
14:00 – 14:05
Introduction, Presentation Of The Workshop & Speakers
MATTHIEU PESANT, PhD, Product Manager NGS, Takara Bio, France

Dr Matthieu Pesant has been the European NGS Product Manager at Takara Bio Europe since 2017 and manages Takara’s RNA-seq and DNA-seq product range including the market leading single-cell NGS technologies SMART-Seq and SMARTer PicoPLEX.

Dr Pesant obtained his PhD in Molecular Biology at the University of Burgundy, France, where he identified regulatory mechanisms of oxidative stress and inflammation relevant to cardiac muscle and atherosclerosis. He then worked as a postdoctoral researcher at the Humanitas Research Center in Milan, Italy, identifying new mechanisms of cellular communication between innate immune cells and cancer cells. He then moved to the National Institute of Molecular Genetics in Milan, Italy, prior to joining Takara Bio Europe, where he contributed to uncover crosstalks between chromatin epigenetic landscapes and genome conformation underlying a genetic muscular dystrophy in human.

14:05 – 14:25

Somatic CNV Detection By Single Cell Whole Genome Sequencing In Multiple System Atrophy, A Sporadic Neurodegenerative Disorder

CHRISTOS PROUKAKIS, PhD, FRCP, Associate Professor, UCL Queen Square Institute of Neurology, and Honorary Consultant Neurologist, Royal Free London NHS Trust, UK | Twitter: @proukakis

Christos Proukakis is a practicing clinical academic neurologist, currently an Associate Professor in the University College London Institute of Neurology, and Honorary Consultant Neurologist at the Royal Free London. His clinical and research focus is Parkinson’s disease / synucleinopathies. He hypothesized and is investigating the role of somatic mutations in the brain. His lab demonstrated somatic copy number gains (CNVs) in the alpha-synuclein gene, and produced the first genome-wide somatic CNV calls in synucleinopathy brain using single cell whole genome sequencing. He is also using long-read sequencing for the difficult GBA gene.
 
Presentation Details:
Synucleinopathies are mostly sporadic neurodegenerative disorders of partly unexplained aetiology, and include Parkinson’s disease (PD) and multiple system atrophy (MSA). We have further investigated our recent finding of somatic SNCA (α-synuclein) copy number variants (CNVs, specifically gains) in synucleinopathies, using Fluorescent in-situ Hybridisation for SNCA, and single-cell whole genome sequencing for the first time in a synucleinopathy. In the cingulate cortex, mosaicism levels for SNCA gains were higher in MSA and PD than controls in neurons (> 2% in both diseases), and for MSA also in non-neurons. In MSA substantia nigra (SN), we noted SNCA gains in > 3% of dopaminergic (DA) neurons (identified by neuromelanin) and neuromelanin-negative cells, including olig2-positive oligodendroglia. Cells with CNVs were more likely to have α-synuclein inclusions, in a pattern corresponding to cell categories mostly relevant to the disease: DA neurons in Lewy-body cases, and other cells in the striatonigral degeneration-dominant MSA variant (MSA-SND). Higher mosaicism levels in SN neuromelanin-negative cells may correlate with younger onset in typical MSA-SND, and in cingulate neurons with younger death in PD. Larger sample sizes will, however, be required to confirm these putative findings. We obtained genome-wide somatic CNV profiles from 169 cells from the substantia nigra of two MSA cases, and pons and putamen of one. These showed somatic CNVs in ~ 30% of cells, with clonality and origins in segmental duplications for some. CNVs had distinct profiles based on cell type, with neurons having a mix of gains and losses, and other cells having almost exclusively gains, although control data sets will be required to determine possible disease relevance. We propose that somatic SNCA CNVs may contribute to the aetiology and pathogenesis of synucleinopathies, and that genome-wide somatic CNVs in MSA brain merit further study.
14:25 – 14:45
Combined Single Cell TCR & Transcriptomics Using The ICELL8
WILFRED VAN IJCKEN, PhD, Head of Center for Biomics, Erasmus MC, Rotterdam, Netherlands
Dr. Wilfred van IJcken (Board Member of Methylomics and Manager of Erasmus Center for Biomics, Erasmus medical Center Rotterdam, The Netherlands) is managing the Genomics Core Facility of Erasmus MC, largest medical center in The Netherlands. He received his PhD in Genomics Virology from Wageningen University & Research Center in 2001. He then joined as a post-doc the group of Prof. Frank Grosveld at Erasmus MC in Rotterdam, where he initiated and started a genomics core facility. In 2007 he was one of the first offering Next Generation Sequencing to the Dutch research community. Dr. ir. van IJcken, who is assistant professor since 2008, has over 200 publications in peer-review journals and his most recent one is about Combined single T-cell sequencing of transcriptome and TCR repertoire profiling in CMV infected samples. His study lead to a better understanding of immunological response to viral infection.
 
Presentation Details:
Transcriptomics can be combined with TRA and TRB clonotype analysis at the single cell level. This presentation shows our validation of this approach on the ICELL8 Single-Cell system and to evaluate its usefulness to analyse clinical paucicellular samples. For this purpose, we carefully selected T cell lines with defined TRA/TRB clonotypes as well as clinical samples enriched for CD3+ T cells that possess a complex TCR repertoire. Low cell numbers of the different samples were dispensed in a chip on the ICELL8 Single-Cell System. Two sequencing libraries were generated from each single cell cDNA preparation, one for the TRA/TRB repertoire and one for the 5′ ends of transcripts, and subsequently sequenced. Results of the single cell transcriptomics and TCR clonotyping will be shown as well as validation results to other technologies. The findings can facilitate future studies to examine the response of single T cells in heterogeneous samples, and lead to improved treatments.

14:45 – 15:05

CD4/CD8 Lineage Choice At The Single Cell Level
PROF. MATTHIAS MERKENSCHLAGER, MRC London Institute of Medical Sciences, Imperial College London, UK

We use lymphocytes and ES cells as models to study how gene expression patterns are established, transmitted through cell division and changed during development

We study transcriptional and epigenetic mechanisms that underlie cellular differentiation and explore mechanisms of experimental reprogramming. Our core research activities include the following interrelated areas:

  • Reactivation of imprinted and X-linked genes in vitro and in vivo
  • Chromosome structure, Pc-G genes and epigenetic inheritance
  • The role of 3D genome organisation in gene expression and disease
  • Mechanisms of gene regulation by Ikaros and other transcription factor families

 

By combining classical cell biology and genetics approaches with current technologies we aim to understand how gene expression patterns are regulated during development as well as the consequences of mis-regulation in disease. This mechanistic information can provide a rationale for therapeutic intervention.

15:05 – 15:25
Dissection Of Gene Expression Heterogeneity At The Single-Cell Resolution In CRISPR-Cas9-Induced Mouse Models Of Acute Erythroid Leukemia
ILARIA IACOBUCCI, PhD, Staff Scientist, Department of Pathology at St. Jude Children’s Research Hospital, Memphis, USA

Dr. Ilaria Iacobucci, PhD, is a Staff Scientist in the Department of Pathology at St. Jude Children’s Research Hospital, Memphis (USA). She graduated in Medical Biotechnology (Faculty of Medicine and Surgery), with honors, at the University of Bologna in 2004. She subsequently obtained her PhD in Clinical and Experimental Hematology (2005‒2008) at the University of Bologna, progressed to Postdoctoral Research Fellow (2008‒2011) and was promoted to a to a faculty position in the Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Italy (2011-2014). Her research focuses on the understanding of mechanisms underlying the pathogenesis of high-risk myeloid and lymphoid leukemia subtypes in both children and adults through the identification of novel genetic alterations with a diagnostic, prognostic and therapeutic relevance and the development of preclinical in-vitro and in-vivo models which faithfully recapitulate human leukemia. She has published >120 peer-reviewed articles and contributed to the identification of multiple new subtypes of acute lymphoblastic leukemia, such as EPOR-rearranged, DUX4/ERG-deregulated, MEF2D-rearranged and PAX5 P80R mutated. She has defined the genomic landscape of acute erythroid leukemia and identified multiple subgroups with distinct genomic features and transcriptional profiles and generated a number of new engineered mouse models of lymphoid, erythroid and myeloid leukemia that faithfully recapitulate the genetic alterations observed in human diseases. She is a recipient of many international awards, including American Association for Cancer Research -GlaxoSmithKline Outstanding Clinical Scholar Award, Lady Tata Memorial Trust International Award Fellowship, ISSNAF (Italian Scientists and Scholars in North America Foundation) Paola Campese Award for Research on leukemia and first place in UNDER40 in Hematology in 2019 from the Italian Society of Hematology. Her research modeling erythroleukemia was presented as Plenary Scientific presentation at the 60th ASH Annual Meeting and her recent large-scale genomic analysis revising the taxonomy of myeloid disease was presented as Late Breaking Abstract presentation at the 61th ASH Annual Meeting.

 
Presentation Details:
Acute erythroid leukemia (AEL) is characterized by distinct morphology, mutational spectrum, a lack of preclinical models and poor prognosis. We have recently defined the genetic and transcriptome landscapes of this disease (Iacobucci I. et al. Nature Genetics 2019) and here we used multiplexed genome editing of mouse hematopoietic stem and progenitor cells to induce combination of loss of function mutations in recurrently mutated genes and develop preclinical models of AEL.  We demonstrated the central role of mutational cooperativity in determining leukemia lineage. Combination of mutations in Bcor, Trp53, Dnmt3a, Rb1 and Nfix specified erythroid phenotype, and were accompanied by the acquisition of alterations in signaling genes and transcription factors that recapitulated the genomic features of human AEL. Bulk transcriptome sequencing and methylation array analysis showed that mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. We showed that fluctuation in mutational clonal composition drives changes in leukemia phenotype. In a triple Bcor/Rb1/Trp53-mutant AEL model, we observed expansion of cells with mixed AEL/B lymphoid phenotype (GATA1+, CD44+, B220+, CD19+, PAX5+) in secondary passages. To investigate phenotypic architecture, we performed full-length RNA-sequencing (SMART-seq, Takara) coupled with high parameter flow cytometric immunophenotyping that identified four gene expression clusters associated to distinct leukemia phenotype. Immature hematopoietic cells with expression of both primitive erythroid and lymphoid markers and progenitor erythroid cells were predominant in the primary tumor, while more differentiated erythroid cells and mixed B-lymphoid/erythroid cells were dominant in the secondary tumor passage. Single-cell RNA seq identified an alternative isoform of Bcor in mixed B/erythroid cells due to a larger Bcor gene deletion. In contrast to the truncated Bcor alleles induced by genome editing in the primary tumor, expression of this BCOR isoform was associated with downregulation of components of non-canonical polycomb repressive complex 1 (PRC1.1) and overexpression of its repressed target genes, including lymphoid markers. Overall, integrated single-cell genomic/transcriptomic profiling directly demonstrated the relationship of initiating and secondary genomic lesions and their relationship to leukemic phenotype.
15:25 – 15:45
Advances In Single-Cell Combinatorial Indexing
ANDREW ADEY, PhD, Associate Professor of Molecular & Medical Genetics, Oregon Health & Science University, USA

Dr. Adey received his undergraduate degree in Biochemistry from the University of Texas where he worked in the lab of Dr. Andrew D. Ellington and served as the interim director of the UT Microarray Core Facility. He then completed his doctoral studies in Molecular and Cell Biology in the lab of Dr. Jay Shendure at the Genome Sciences Department at the University of Washington. During his graduate studies he pioneered several DNA sequencing technologies, including some that are still routinely used around the globe. He also led work to sequence and characterize the genome of the HeLa cancer cell line where he utilized new technologies to provide novel insights into gene regulation in cancer. After completing his graduate studies he started his own independent group in the Department of Molecular and Medical Genetics at the Oregon Health and Science University, where he is now an Associate Professor. He is also a member of the Knight Cancer Institute, the Cancer Early Detection Advanced Research center, and the Knight Cardiovascular Institute. He was also the recipient of the 2018 American Society of Human Genetics Early Career Award. The Adey Lab is currently focused on the development and deployment of single-cell technologies to profile a variety of epigenetic properties with an emphasis on chromatin accessibility as well as the spatial tracking of cell positions. These technologies are being deployed to better understand fundamentals of gene regulation and their implications in neurodevelopment and cancer.

Presentation Details:
Single-cell technologies have revolutionized how we approach our study of complex biological systems. We have developed a platform for assaying several properties at the single-cell level in high throughput using combinatorial indexing methods, where cells are barcoded multiple times. In order to scale up the number of cell profiles that can be produced without sacrificing data quality, we have increased the index space at each round of cell barcoding with the aid of nanowell chips to enable hundreds of thousands of high quality cell profiles to be generated in a single low-cost experiment.

15:45 – 16:35

Expert Panel Discussion:
Panel formed of all workshop speakers
16:35 – 16:55
Single Cells In Health & Disease
ANDREW FARMER, PhD, CSO, Takara Bio, USA

Andrew obtained his undergraduate degree in physiology from the University of Oxford. He stayed on at Oxford for his doctoral work, researching the control of liver differentiation with Dr. Stephen Goss. After coming to the USA, he did post-doctoral research in the field of tumor suppressor genes, working first with Professor Eric Stanbridge and later with Dr. Wen-Hwa Lee.

Andrew joined Takara Bio USA (TBUSA: formerly Clontech) in 1998 as the developing scientist for the Tet-Systems and was also responsible for inventing a recombination-based cloning technology known as the Creator System. Over the years, he has been responsible for the development of several of Clontech’s product lines, including: Two-hybrid systems, viral delivery and inducible expression systems, and the In-Fusion Cloning system. Most recently, Andrew lead TBUSA’s development of its NGS product line – most notably its suite of ultra-low input RNA-Seq products based on TBUSA’s SMART technology. Currently, he is TBUSA’s CSO / Head of R&D.  

Presentation Details:
From small beginnings a little over a decade ago, since cell genomics has now truly come of age.  An array of platforms and methods allow for high-through put analysis of thousands – even millions – of cells across a range of modalities including gene expression, epigenetics and even genomics.   The age of consortia – include the Human Cell Atlas, LifeTime and HuBMAP – now seek to apply these technologies to the understanding of human physiology and pathology, building on the groundwork laid by the Human Genome Project. This talk will briefly summarize how single cell genomics is expanding our understanding of both health and disease.

16:55 – 17:00
Wrap Up & Conclusion
MATTHIEU PESANT, PhD, Product Manager NGS, Takara Bio, France