Cellular heterogeneity plays a critical role in tissues and diseases, including cancer. Single-cell technologies are required to provide detailed information about the phenotype and genotype of individual cells. Despite several approaches to analyzing different analytes at the single-cell level, it is challenging to assess DNA, RNA, and protein simultaneously. Here, a single-cell triomics method to assess DNA, RNA, and proteins from the same cell using a targeted sequencing approach is shown. Breast cancer cells cultured in monolayers and in patient-derived scaffolds that mimic in vivo-like growth conditions, both with and without chemotherapy treatment, were analyzed. Data showed that DNA, RNA, and protein biomarkers could be reliably analyzed, providing biological insights into breast cancer cell heterogeneity. In addition, chemotherapy treatment caused changes in subpopulations and expressions of biomarkers. Furthermore, cells growing in patient-derived scaffolds generated from various breast cancers affected cell heterogeneity and drug resistance differently as a result of the unique tumor-specific microenvironments. The data show that single-cell triomics provides new means to assess cancer cell heterogeneity at DNA, RNA, and protein levels.

We provide an overview of activities carried out at the TJ-II stellarator for improving our understanding of- and developing plasma physics models for particle density profiles in stellarators. Namely, we report on recent progress in turbulent particle transport simulation, validation of pellet deposition models, density profile shaping for performance control and new experimental techniques for edge turbulence and plasma-neutral interaction.

The tremendous advances in genetic studies owe a great deal to the completion of human genome project in 2003 and the availability of next-generation sequencing (NGS). Illumina has the biggest NGS market share worldwide. Since Illumina acquired Solexa in 2007, Illumina has led NGS research and applications. Illumina has released many different types of platforms, from a small scale sequencer to a highest throughput sequencer. The first commercialized NGS system shown on the market was 454 pyrosequencing in 2005. For this system, dNTPs are added to the template-bound bead in a PicoTiterPlate. When each dNTP is incorporated into a newly synthesized DNA strand, a pyrophosphate molecule is produced and reacted with adenosine triphosphate (ATP) sulfurylase, leading to a change of adenosine phosphosulfate into ATP. The covered ATP with luciferase changes luciferin into light and oxyluciferin. The chapter discusses several different NGS or third-generation sequencing platforms and sample preparation methods.

Supplementary Figures S1-S3; Supplementary Tables S1-S14.

The International Fusion Materials Irradiation Facility (IFMIF) is a project aiming to investigate candidate materials to be used in the most exposed zones of future fusion reactors. The linear IFMIF prototype accelerator (LIPAc), presently under commissioning in Rokkasho, Japan, is a prototype of the frontend section of one of the IFMIF accelerators. Eight quadrupole magnets, six pairs of corrector magnets and one dipole are responsible for generating the magnetic fields needed for a proper beam handling along the 10 m long LIPAc high energy beam transport line, which connects the end of the superconducting radio frequency Linac with the beam dump. A novel design of combined magnets with the correctors integrated in the quadrupole poles is chosen for compactness reasons. The different stages of the production of the combined magnets, from the magnetic and mechanical design to their manufacturing and testing, including exhaustive characterization of the magnetic performance are described in this work. The results from the tests revealed the quality of the magnetic field produced. The materials selection was done carefully, to withstand the high levels of ionizing radiation expected at the magnet locations. This paper focuses on the activities performed in Europe, before sending the magnets to Japan for their installation and commissioning at the Rokkasho site.

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

TJ-II stellarator results on modelling and validation of plasma flow asymmetries due to on-surface potential variations, plasma fuelling physics, Alfvén eigenmodes (AEs) control and stability, the interplay between turbulence and neoclassical (NC) mechanisms and liquid metals are reported. Regarding the validation of the neoclassically predicted potential asymmetries, its impact on the radial electric field along the flux surface has been successfully validated against Doppler reflectometry measurements. Research on the physics and modelling of plasma core fuelling with pellets and tracer encapsulated solid pellet injection has shown that, although post-injection particle radial redistributions can be understood qualitatively from NC mechanisms, turbulence and fluctuations are strongly affected during the ablation process. Advanced analysis tools based on transfer entropy have shown that radial electric fields do not only affect the radial turbulence correlation length but are also capable of reducing the propagation of turbulence from the edge into the scrape-off layer. Direct experimental observation of long range correlated structures show that zonal flow structures are ubiquitous in the whole plasma cross-section in the TJ-II stellarator. Alfvénic activity control strategies using ECRH and ECCD as well as the relation between zonal structures and AEs are reported. Finally, the behaviour of liquid metals exposed to hot and cold plasmas in a capillary porous system container was investigated.

Advances in microfluidic droplet technologies have greatly facilitated breakthroughs in cellular and biomedical sciences, particularly in the arena of single-cell genomic, transcriptomic and proteomic analysis applications. Microfluidic devices enable the high-throughput preparation of biological samples that are readily partitioned into nanoliter-scale droplets containing isolated individual cells or nuclei. Aqueous droplets are suspended and isolated in a hydrophobic carrier liquid. Typical microfluidic workflows offer the magnitude of multiple thousands upward to millions of encapsulated droplets per experimental run. Biochemical reactions provide for the ready interrogation of cellular components such as genomic DNA, RNA transcripts and protein markers. Innovative tagging and nucleic acid amplification workflows generate droplet-specific barcode identifiers compatible with next-generation sequencing platforms. After barcoding, thousands of droplets are combined together, amplified offline, size purified and sequenced in massive parallel protocols. One such enabling platform is the Mission Bio Tapestri® system. This unique system offers a novel two-stage droplet workflow that provides multiple sequential cascades of enzymatic and chemistry processes, and opens opportunities for multimodal investigations of single cells. We describe Tapestri applications including workflows, reaction overviews and data examples. These microfluidic applications have propelled the frontiers of cellular analysis to further multidimensional analysis of cell velocities in dynamic biological systems.

The International Fusion Materials Irradiation Facility (IFMIF) is a projected accelerator-based, D-Li neutron source for fusion reactor materials qualification. LIPAc (Linear IFMIF Prototype Accelerator) is an accelerator aiming to generate a 125 mA, 9 MeV continuous wave deuteron beam, which is currently being commissioned in Rokkasho (Japan) with the objective of validating the IFMIF accelerator design. In LIPAc, a 10 m long High Energy Beam Transport line (HEBT) will connect the exit of the superconducting linac to the beam dump (BD). The HEBT line must accommodate the diagnostics for beam characterization and open the beam at the end to allow its stopping at the BD. The line contains several magnets to control the beam shape and its trajectory, maintaining beam losses below 1 W m−1 along the beamline to limit activation of surrounding elements and allow hands-on maintenance. In this work, the LIPAc HEBT line project is described since its origins. A summary of the beam dynamics calculations and other studies (vacuum, radioprotection, assembly, alignment) that led to the conceptual design of the line is done. After that, the detailed design of the line is presented, justifying the main design decisions taken and finally, the manufacturing and procurement process and the acceptance tests performed are summarized.

Single-cell technologies are now able to provide information into genomic DNA content, RNA expression, and protein surface markers, unmasked by the heterogeneity found in bulk data. However, multimodal analysis from the same single cell has not been straightforward to implement in a high throughput single-cell workflow. We report the development of chemistries that enable analyses of both targeted genomic DNA and RNA sequencing from the same cell. This workflow relies on the Tapestri platform, which uses a two-step microfluidic droplet system for the analysis of thousands of cells per run. The first droplet encapsulates each cell and releases the DNA while the second droplet introduces additional reagents for enzymatic manipulations on the cellular analytes. Novel primer designs are leveraged to capture both DNA and RNA and provide for independent barcoded sequence information. We first establish the utility of the Tapestri platform for accurate gene expression assessment with a targeted panel for breast cancer. We then demonstrate concordant RNA and DNA reads from single cells with fusion gene transcript detection. A panel was designed for acute myeloid leukemia targeted 20 genes and including primers for multiple variants of BCR-ABL transcripts. Cell lines were used to show high sensitivity and specificity of fusion sequence calls from RNA in cells where the expected SNVs, indels, and CNVs were also detected from gDNA. More complex targeted RNA and DNA panels, such as one for breast cancer with 35 gene expression amplicons and 88 genotyping amplicons, were also tested where we show agreement between cells clustered based on RNA expression and the cell assignment based on SNVs from gDNA. This single-cell multimodal workflow on the Tapestri platform currently has the power to quantitatively link genotypic and phenotypic data with future potential in biomarker identification and ultimately, to link to therapeutics.

Abstract Fusion gene detection has long been a focus of cancer research, when combined with mutations found in gDNA, can lead to a better understanding of disease progression. One example, BCR-ABL, a marker for CML and AML stem cells, is a target for tyrosine kinase inhibitor (TKI) treatments; however, there are mutations within BCR-ABL that evade TKIs and are selectively resistant to drug therapy. Single-cell technologies are now able to provide information into genomic DNA content, RNA expression, and protein surface markers, unmasked by the heterogeneity found in bulk data. However, multimodal analysis from the same single cell has not been straightforward to implement in a high throughput single-cell workflow. Here, we report the development of chemistries that enable analyses of both targeted genomic DNA and RNA sequencing from the same cell. This workflow relies on the Tapestri platform, which uses a two-step microfluidic droplet system for the analysis of thousands of cells per run. The first droplet encapsulates each cell and releases the DNA while the second droplet introduces additional reagents for enzymatic manipulations on the cellular analytes. Novel primer designs are leveraged to capture both DNA and RNA and provide for independent barcoded sequence information. We first establish the utility of the Tapestri platform for accurate gene expression assessment with a targeted panel for breast cancer. For fusion gene transcript detection, panels were designed for AML and CML including primers targeting multiple potential variants of BCR-ABL transcripts. Cell lines were used to show high sensitivity and specificity of fusion sequence calls from RNA in cells where the expected SNVs, indels, and CNVs were also detected from gDNA. More complex targeted RNA and DNA panels, such as one for breast cancer with 35 gene expression amplicons and 88 genotyping amplicons, were also tested where we show agreement between cells clustered based on RNA expression and the cell assignment based on SNVs from gDNA. This single-cell multimodal workflow on the Tapestri platform currently has the power to quantitatively link genotypic and phenotypic data from the same cell with future potential in biomarker identification and ultimately, to link to best-fit therapeutics. Citation Format: Dalia Dhingra, Pedro Mendez, Aik Ooi, Shu Wang, Saurabh Gulati, Adam Sciambi, Dave Ruff. A high throughput single-cell workflow for paired genomic and phenotypic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 219.

Abstract Recent advancements in precision medicine, while highly promising, presents a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multiomic analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights on cellular and genetic composition. Using a two-step droplet microfluidic technology, the Mission Bio Tapestri Platform enables multiplex-PCR based high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a new workflow is developed to detect protein expression in addition to DNA genotype in the same single cells. In this approach, cells are labeled with a pool of oligonucleotide-conjugated antibodies prior to loading the cells into the Tapestri Instrument for targeted DNA analysis. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. In a mixed population of four cell lines, single-cell SNV and CNV information from 127 targeted amplicons and the protein data from 10 antibodies independently classified the cells into appropriate clusters. This method has been successfully performed on clinical samples with myeloid malignancies. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data clearly identified CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs. CD34hiCD11blo and CD34loCD11bhi subpopulations were also identified within the cells carrying the same pathogenic SNVs and CNVs. We believe that this novel multiomic technology will enable new discoveries in the complex relationship between genotype and phenotype, leading to a better understanding of disease biology, and subsequently better design of diagnostics and therapies. Citation Format: Aik Ooi, Pedro Mendez, Dalia Dhingra, Nigel Beard, David Ruff. Single-cell multiomic analysis of SNV, CNV, and protein expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5910.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1–3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression. The evolution of myeloid malignancies is investigated using combined single-cell sequencing and immunophenotypic analysis.

Abstract The TCGA, ICGC and other research consortiums has shown that an integral, multi-omic analysis of tumors is necessary in order to understand a more complete picture of the signaling pathways involved in development, evolution, prognosis and treatment resistance of tumors. However, in such studies, because different data modalities (“omics”) are analyzed separately, the information on how the co-existence of in the same cell is lacking. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using high-throughput droplet microfluidics on the Tapestri platform, paired with next generation sequencing. Our triomic methodology evaluates targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations (SNV & CNV) from the same cell. We employ oligonucleotide-conjugated antibody panels to probe cellular surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with cocktails of oligonucleotide-antibodies. These cells are loaded onto the Mission Bio Tapestri® microfluidic cartridge for generation of the first droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA from chromatin scaffolding. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced and demultiplexed bioinformatically yielding a multi-omics readout from the single-cells. Six protein surface markers, 35 transcripts and 88 genomic regions in 68 genes were interrogated with a 50:50 cell mixture of MCF7 and GM12878 cells. These targets play key roles in cellular migration, stemness, differentiation and proliferation. We intend to further explore the relationship between genotype-to-phenotype in breast carcinoma cell lines cultured in patient-derived scaffolds mimicking tumor microenvironments under different drug regimen conditions. We report the dynamic multimodal resolution and demonstrate utility of triomics in further illuminating cellular states and biological responses. Citation Format: Pedro Mendez, Dalia Dhingra, Aik Ooi, Shu Wang, Saurabh Gulati, Nigel Beard, Manimozhi Manivannan, Jens Björkman, Mikael Kubista, Göran Landberg, David Ruff, Anders Ståhlberg. Simultaneous DNA, RNA and protein analysis from single cells using a high-throughput microfluidic workflow for resolution of genotype-to-phenotype modalities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2506.

Summary Myeloid malignancies, including acute myeloid leukemia (AML), arise from the proliferation and expansion of hematopoietic stem and progenitor cells which acquire somatic mutations. Bulk molecular profiling studies on patient samples have suggested that somatic mutations are obtained in a step-wise fashion, where mutant genes with high variant allele frequencies (VAFs) are proposed to occur early in disease development and mutations with lower VAFs are thought to be acquired later in disease progression 1–3 . Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity and clone size, or offer definitive evidence of mutational order. To elucidate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is most commonly comprised of a small number of dominant clones, which in many cases harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We also used these data to map the clonal trajectory of each patient and found that specific mutation combinations ( FLT3-ITD + NPM1 c ) synergize to promote clonal expansion and dominance. We combined cell surface protein expression with single cell mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of clonal architecture at a single cell level provide novel insights into the pathogenesis of myeloid transformation and how clonal complexity contributes to disease progression.

In the framework of the IFMIF/EVEDA project, the cryomodule of the Linear IFMIF Prototype Accelerator (LIPAc) will be assembled then tested at Rokkasho in 2019. Eight Series Power Couplers (PC) operating at 175 MHz were manufactured under a CEA contract, in order to equip this Cryomodule. They were all successfully RF conditioned up to 100 kW CW in TW and SW configurations. All the high RF power tests were performed under CIEMAT responsibility in BTESA Company premises, according to the CEA requirements. In order to fix difficulties encountered during the fab process, manufacturing and quality control have been analyzed in depth. Thanks to the corrective actions implemented, every PC reached the performances targeted for qualification. This paper will give details about this manufacturing phase and provide an overview of the obtained RF test results.

Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by H&E-staining. Many human samples provide outstanding quality nuclei suspensions, including cryopreserved cell lines, PBMCs, bone marrow, liver, brain, breast, colon, lung, prostate and melanomas. Fixed and preserved specimens also yield nuclei readily interrogated by targeted DNA sequencing panels. Nuclei suspensions are readily processed with the Tapestri Platform for single-cell DNA analysis. Leveraging droplet microfluidics and barcoding, the workflow enables high uniformity of ~90%, low ADO of ~10%, and typical nuclei recovery rates exceed 10%. Up to 20,000 nuclei can be interrogated in each run with catalog or custom panels for any tumor type. Here, a 59-gene tumor hotspot panel was used to study five melanoma metastases. The single-cell data enabled the unique reconstruction of tumor sample clonal phylogeny unresolved by bulk analysis. Also, low prevalence metastatic subclones masked in bulk NGS data were detected in normal liver samples. In summary, we show that diverse types of archived tumor tissues are readily dissociated with this universal nuclei protocol. Researchers now have a highly sensitive, targeted, customizable solution for revealing genomic variation and clonal propagation in complex archived solid tumor samples.Citation Format: David Ruff, Pedro Mendez, Daniel Mendoza, Nianzhen Li, Adam Sciambi, Kaustubh Gokhale, Dalia Dhingra, Keith Jones, Dennis Eastburn. High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platform. Viable cells were sorted from 90 samples from 78 patients with CH, AML, and MPN/post-MPN AML followed by single cell amplification/sequencing. Mutation calls were filtered based on read depth, quality, and alleles genotyped per cell. We reconstructed a random distribution of clones by permuting genotype calls across cells and generated empirical p values for each clone. To identify dominant clones, we used a Poisson test to determine clones were significantly enriched compared to the mean clone size. Clones with significant p-values (p <0.05) were used to generate plots of clonal architecture of each sample (Figure 1A). Despite significant clonal complexity, the majority of MM patients (80%;72/90) present with one (51/90; 56.7%) or two (21/90; 23.3%) dominant clones. These data show there are specific genotypic combinations which lead to clonal dominance with increased fitness relative to other clones and/or suppression of minor clones by dominant clone(s). We next investigated whether specific molecularly defined AML subtypes had increased clonal complexity. FLT3-ITD mutant AML samples had a significantly greater number of clones (p < 0.002) compared to AML samples with multiple epigenetic modifier mutations. Similar findings were not observed when comparing AML samples with epigenetic mutations to RAS pathway mutant samples. We next investigated whether specific mutations were likely to co-occur/be mutually exclusive at a single cell level. We observed evidence of oligoclonality in CH, including parallel acquisition of DNMT3A mutations and clones with multiple mutations in the absence of progression to MM. By contrast, in MM the dominant clone(s) almost always harbored multiple epigenetic modifier mutations, suggesting cooperative epigenetic remodeling in myeloid transformation. Mutations in signaling effectors (FLT3-ITD/TKD; RAS/RAS) were mutually exclusive. We observed distinct FLT3-mutant clones in FLT3-mutant AML patients and parallel acquisition of different RAS pathway mutations. We used this data to develop clonal architecture trees in each patient, giving us a definitive picture of mutational acquisition and transformation at a single cell level. We calculated a Shannon diversity score and observed an increase in clonal complexity with disease evolution; CH samples had the lowest clonal diversity and FLT3-ITD AML patients the highest clonal diversity (Figure 1B). We extended our findings by combining cell surface marker assessment and single cell mutational analysis. Patient samples were stained with an antibody cocktail of 6 oligo-conjugated antibodies with barcode tags prior to single cell sequencing, which allowed simultaneous acquisition of single cell immunophenotypic and genotypic data. This allows us to identify distinct populations of stem/progenitor cells with distinct clonal/mutational repertoires (Figure 1C). Additional data will be presented with this novel approach, which allows us to combine an assessment of stem/progenitor cell frequency with genetic data. This includes studies of CD34+ and CD34- AML, which show striking differences in mutational representation in different stem/progenitor compartments. In summary, our studies of clonal architecture at a single cell level provide us novel insights into the pathogenesis of myeloid transformation and give us new insights into how clonal complexity contributes to disease progression. Disclosures Ooi: Mission Bio: Employment, Equity Ownership. Mendez:Mission Bio: Employment, Equity Ownership. Carroll:Janssen Pharmaceuticals: Consultancy; Incyte: Research Funding; Astellas Pharmaceuticals: Research Funding. Papaemmanuil:Celgene: Research Funding. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Roche: Consultancy, Research Funding; Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Gilead: Consultancy; Celgene: Consultancy, Research Funding.

The main results obtained in the TJ-II stellarator in the last two years are reported. The most important topics investigated have been modelling and validation of impurity transport, validation of gyrokinetic simulations, turbulence characterisation, effect of magnetic configuration on transport, fuelling with pellet injection, fast particles and liquid metal plasma facing components. As regards impurity transport research, a number of working lines exploring several recently discovered effects have been developed: the effect of tangential drifts on stellarator neoclassical transport, the impurity flux driven by electric fields tangent to magnetic surfaces and attempts of experimental validation with Doppler reflectometry of the variation of the radial electric field on the flux surface. Concerning gyrokinetic simulations, two validation activities have been performed, the comparison with measurements of zonal flow relaxation in pellet-induced fast transients and the comparison with experimental poloidal variation of fluctuations amplitude. The impact of radial electric fields on turbulence spreading in the edge and scrape-off layer has been also experimentally characterized using a 2D Langmuir probe array. Another remarkable piece of work has been the investigation of the radial propagation of small temperature perturbations using transfer entropy. Research on the physics and modelling of plasma core fuelling with pellet and tracer-encapsulated solid-pellet injection has produced also relevant results. Neutral beam injection driven Alfvénic activity and its possible control by electron cyclotron current drive has been examined as well in TJ-II. Finally, recent results on alternative plasma facing components based on liquid metals are also presented.

Recent advancements in single cell analysis technologies are now able to provide insights into genomic DNA content, RNA expression and protein surface markers. In bulk assays, the effect of genetic variation on gene expression would be masked by the heterogeneity inherent in tumor cells. Additionally, for cancer immunotherapy studies that rely on gene editing, single cell resolution is necessary to minimize possible off target effects. However, the ability to simultaneously interrogate multiple intracellular analytes, such as genomic DNA and RNA, have proved difficult to implement in a high throughput single cell workflow. We report the development of a complete solution that enables both targeted genomic DNA and RNA sequencing from individual cells. This workflow relies on the Tapestri microfluidic droplet platform, where up to 20,000 cells can be sequenced in each run. Leveraging proprietary cell barcoding, novel primer design strategies and enzymatic manipulation of cellular contents, DNA and RNA multiplex targeted sequencing panels provide for independent barcoded sequence information from both overlapping mRNA and corresponding genomic DNA regions. In addition, amplification primers can be designed to target separate gDNA and non-overlapping RNA transcript. Sequencing is followed by an integrated analysis solution that assigns the reads from both the gDNA and RNA to each cell. Feasibility of the targeted nucleic acid workflow has been shown with inputs from mixed cancer cell lines. A targeted sequencing panel with overlapping mRNA and gDNA regions was designed covering oncogenes, tumor suppressor genes, and known fusions. Expected SNVs and indels were detected and gene expression measured for thousands of cells per run with high cell recovery. This complete solution for single cell multiomics on the Tapestri platform has the power to quantitatively and unambiguously link genotypic and phenotypic data, giving insight into cancer progression. Citation Format: Dalia Dhingra, Kaustubh Gokhale, Nianzhen Li, Pedro Mendez, Shu Wang, Manimozhi Manivannan, Adam Sciambi, Keith Jones, Charlie Silver, Dennis Eastburn, David Ruff. A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3540.