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Multimodal data is rapidly growing in many fields of science and engineering, including single-cell biology. We introduce MultiMAP, a novel algorithm for dimensionality reduction and integration. MultiMAP can integrate any number of datasets, leverages features not present in all datasets, is not restricted to a linear mapping, allows the user to specify the influence of each dataset, and is extremely scalable to large datasets. We apply MultiMAP to single-cell transcriptomics, chromatin accessibility, methylation, and spatial data and show that it outperforms current approaches. On a new thymus dataset, we use MultiMAP to integrate cells along a temporal trajectory. This enables quantitative comparison of transcription factor expression and binding site accessibility over the course of T cell differentiation, revealing patterns of expression versus binding site opening kinetics.

Psoriatic arthritis (PsA) is a debilitating immune-mediated inflammatory arthritis of unknown pathogenesis commonly affecting patients with skin psoriasis. Here we use complementary single-cell approaches to study leukocytes from PsA joints. Mass cytometry demonstrates a 3-fold expansion of memory CD8 T cells in the joints of PsA patients compared to peripheral blood. Meanwhile, droplet-based and plate-based single-cell RNA sequencing of paired T cell receptor alpha and beta chain sequences show pronounced CD8 T cell clonal expansions within the joints. Transcriptome analyses find these expanded synovial CD8 T cells to express cycling, activation, tissue-homing and tissue residency markers. T cell receptor sequence comparison between patients identifies clonal convergence. Finally, chemokine receptor CXCR3 is upregulated in the expanded synovial CD8 T cells, while two CXCR3 ligands, CXCL9 and CXCL10, are elevated in PsA synovial fluid. Our data thus provide a quantitative molecular insight into the cellular immune landscape of psoriatic arthritis. Psoriatic arthritis (PsA) commonly affects patients with skin psoriasis, but its pathogenesis is still unclear. Here the authors use two types of single-cells data, mass cytometry and RNA sequencing, to describe the expansion and diversity of synovial, but not peripheral blood, CD8 T cells from PsA patients to provide a molecular immune landscape for PsA.

Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit–based workflow. The protocols take 3–5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative ( www.humancellatlas.org ). In this protocol, the authors describe two automated versions of the Smart-seq2 method for full-length single-cell RNA sequencing: a medium-throughput variant using off-the-shelf reagents and a high-throughput version using a commercially available kit.

Matthew Hurles and colleagues report the sequencing of breakpoints for over 300 CNVs detected in genomes of three individuals, using targeted hybridization-based DNA capture and 454-sequencing. Precisely characterizing the breakpoints of copy number variants (CNVs) is crucial for assessing their functional impact. However, fewer than 10% of known germline CNVs have been mapped to the single-nucleotide level. We characterized the sequence breakpoints from a dataset of all CNVs detected in three unrelated individuals in previous array-based CNV discovery experiments. We used targeted hybridization-based DNA capture and 454 sequencing to sequence 324 CNV breakpoints, including 315 deletions. We observed two major breakpoint signatures: 70% of the deletion breakpoints have 1−30 bp of microhomology, whereas 33% of deletion breakpoints contain 1−367 bp of inserted sequence. The co-occurrence of microhomology and inserted sequence is low (10%), suggesting that there are at least two different mutational mechanisms. Approximately 5% of the breakpoints represent more complex rearrangements, including local microinversions, suggesting a replication-based strand switching mechanism. Despite a rich literature on DNA repair processes, reconstruction of the molecular events generating each of these mutations is not yet possible.

Positional coding along the anterior-posterior axis is regulated by HOX genes, whose 3’ to 5’ expression correlates with location along this axis. The precise utilisation of HOX genes in different human cell types is not fully understood. Here, we use single-cell and spatial-transcriptomics, along with in-situ sequencing, to create a developmental atlas of the human fetal spine. We analyse HOX gene expression across cell types during development, finding that neural-crest derivatives unexpectedly retain the anatomical HOX code of their origin while also adopting the code of their destination. This trend is confirmed across multiple organs. In the axial plane of the spinal cord, we find distinct patterns in the ventral and dorsal domains, providing insights into motor pool organisation and loss of collinearity in HOXB genes. Our findings shed new light on HOX gene expression in the developing spine, highlighting a HOX gene ‘source code’ in neural-crest cell derivatives. The HOX gene cluster is responsible for anteroposterior axis patterning in an evolutionarily conserved manner. Here they examine HOX gene expression in human embryos and show that neural-crest derivatives retain the anatomical HOX code of their origin while also adopting the code of their destination.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora- knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora , most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

By using a reverse transcriptase for the bridge-amplification step on the Illumina Genome Analyzer, RNA conversion to cDNA and sequencing take place directly in the flowcell and yield highly accurate strand-specific sequences. We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A) + RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.

Background Sequencing of the genome of Propionibacterium acnes produced a catalogue of genes many of which enable this organism to colonise skin and survive exposure to the elements. Despite this platform, there was little understanding of the gene regulation that gives rise to an organism that has a major impact on human health and wellbeing and causes infections beyond the skin. To address this situation, we have undertaken a genome–wide study of gene regulation using a combination of improved differential and global RNA-sequencing and an analytical approach that takes into account the inherent noise within the data. Results We have produced nucleotide-resolution transcriptome maps that identify and differentiate sites of transcription initiation from sites of stable RNA processing and mRNA cleavage. Moreover, analysis of these maps provides strong evidence for ‘pervasive’ transcription and shows that contrary to initial indications it is not biased towards the production of antisense RNAs. In addition, the maps reveal an extensive array of riboswitches, leaderless mRNAs and small non-protein-coding RNAs alongside vegetative promoters and post-transcriptional events, which includes unusual tRNA processing. The identification of such features will inform models of complex gene regulation, as illustrated here for ribonucleotide reductases and a potential quorum-sensing, two-component system. Conclusions The approach described here, which is transferable to any bacterial species, has produced a step increase in whole-cell knowledge of gene regulation in P. acnes . Continued expansion of our maps to include transcription associated with different growth conditions and genetic backgrounds will provide a new platform from which to computationally model the gene expression that determines the physiology of P. acnes and its role in human disease.

It is not fully understood why COVID-19 is typically milder in children 1 – 3 . Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n  = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children. Mechanisms explaining the milder clinical syndrome that is observed in children with SARS-CoV-2 infection.