Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human’s genome is edited by these two ...processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual’s gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.
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► A microfluidic system for high-throughput single cell whole-genome amplification ► Personal recombination map by single-sperm whole-genome analysis ► Genome-wide meiotic drive and gene conversion tests ► De novo germline mutations with distinct molecular characteristics
A new high-throughput method, based on a microfluidic system, generates a personal recombination map from 91 individual sperm cells. The single-cell sequencing data provide measurements of gene conversion activity and genome instability rates in human sperm.
The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human ...cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years.
Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their ...transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5−/− cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.
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•Single-cell RNA-seq uncovers chamber-specific genes in the embryonic mouse heart•Machine learning can infer anatomical context from single-cell transcriptional data•Nkx2-5−/− embryonic mouse cardiomyocytes lack a ventricular transcriptional profile•Embryonic ventricular myocardium display trabecular-compact expression gradients
Cardiogenesis is orchestrated by cell-type- and chamber-specific transcription. Li et al. collected 2,233 single-cell RNA-seq samples from embryonic mouse hearts. This data resource uncovers anatomical patterns of gene expression that enable the deduction of a single-cell sample's anatomical origin, providing insight into developmental perturbations in congenital heart defect models.
Significance Latent viral infection is a major obstacle for effective antiviral treatment and presents a continuous risk to the host. The dormant viral genome during latent infection provides few ...therapeutic targets other than itself for antiviral drug development. This study demonstrates the clearance of latent Epstein–Barr virus genomes in a subpopulation of Burkitt’s lymphoma patient-derived cells with clustered regularly interspaced short palindromic repeat/Cas9 nuclease. Viral genome destruction leads to proliferation arrest and apoptosis in Epstein–Barr virus-infected cells, with no observed cytotoxicity to noninfected cells. Although many hurdles remain before this approach could be used in the clinic, this strategy may lead to a generalized approach to cure latent viral infections.
There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ ...transplant recipients are treated with posttransplant therapies that combine immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients) and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment and offer a potential application of the virome state to predict immunocompetence.
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•Virome-drug interactions were measured in a cohort of organ transplant recipients•The structure of the virome is strongly affected by immune modulation and antivirals•The total viral load increases markedly at the onset of the therapy•A potential application of the virome state in predicting immune strength is reported
Using DNA sequencing of cell-free DNA extracted from plasma of organ transplant recipients, this paper investigates the response of the virome, the viral component of the microbiome, to the strong immune modulations.
Many cancers have substantial genomic heterogeneity within a given tumor, and to fully understand that diversity requires the ability to perform single cell analysis. We performed targeted sequencing ...of a panel of single nucleotide variants (SNVs), deletions, and IgH sequences in 1,479 single tumor cells from six acute lymphoblastic leukemia (ALL) patients. By accurately segregating groups of cooccurring mutations into distinct clonal populations, we identified codominant clones in the majority of patients. Evaluation of intraclonal mutation patterns identified clone-specific punctuated cytosine mutagenesis events, showed that most structural variants are acquired before SNVs, determined that KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and identified clones within the same patient that are arrested at varied stages in B-cell development. Taken together, these data order the sequence of genetic events that underlie childhood ALL and provide a framework for understanding the development of the disease at single-cell resolution.
As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic ...process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.
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•RNA-seq of single cells from donors allows detection of stochastic age-related errors•Cells from older donors have increased transcriptional noise and signs of fate drift•Endocrine pancreas cells display an oxidative stress-related mutational signature•Cellular stress and metabolic genes are high in cells with accumulation of errors
Aging is associated with increased transcriptional dysregulation and loss of identity at the single-cell level
Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of ...methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.
In recent years there has been tremendous progress towards deep molecular characterization of cell types using single-cell transcriptome sequencing, creating so-called 'cell atlases'. These atlases ...provide a basic understanding of how different cell types of the same organism - which all share the genome - make distinct use of subsets of genes from the genome to create a variety of distinct cell types across tissues with specialized functions. In this opinion article I discuss some of the history and technological innovations that led to the development of whole-organism atlases.
The cell as a bag of RNA Quake, Stephen R.
Trends in genetics,
December 2021, 2021-12-00, 20211201, Letnik:
37, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Genomic sequencing has provided insight into the genetic characterization of many organisms, and we are now seeing sequencing technologies turned towards phenotypic characterization of cells, ...tissues, and whole organisms. In particular, single-cell transcriptomic techniques are revolutionizing certain aspects of cell biology and enabling fundamental discoveries about cellular diversity, cell state, and cell type identity. I argue here that much of this progress depends on abstracting one’s view of the cell to regard it as a ‘bag of RNA’.
Single-cell transcriptomics techniques enable one to abstract the cell as if it were a ‘bag of RNA’.Although this simplification neglects many important structural aspects of the cell, it enables profound insight into the nature of cell state and cell identity.Cell atlases derived by these techniques are companions to the genome which help explain cellular phenotype.
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