Concurrent analysis of genome and transcriptome in one single cell.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps-(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus.

Negative Selection Allows DNA Mismatch Repair-Deficient Mouse Fibroblasts to Tolerate High Levels of Somatic Mutations.

Substantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification.

Analyzing somatic mutations by single-cell whole-genome sequencing.

Somatic mutations are the cause of cancer and have been implicated in other, noncancerous diseases and aging. While clonally expanded mutations can be studied by deep sequencing of bulk DNA, very few somatic mutations expand clonally, and most are unique to each cell. We describe a detailed protocol for single-cell whole-genome sequencing to discover and analyze somatic mutations in tissues and organs.

Somatic mutation burden in relation to aging and functional life span: implications for cellular reprogramming and rejuvenation.

The accrual of somatic mutations has been implicated as causal factors in aging since the 1950s. However, the quantitative analysis of somatic mutations has posed a major challenge due to the random nature of de novo mutations in normal tissues, which has limited analysis to tumors and other clonal lineages. Advances in single-cell and single-molecule next-generation sequencing now allow to obtain, for the first time, detailed insights into the landscape of somatic mutations in different human tissues and cell types as a function of age under various conditions.

Concurrent analysis of genome and transcriptome in one single cell.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps - 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus.

Mitigating age-related somatic mutation burden.

Genomes are inherently unstable and require constant DNA repair to maintain their genetic information. However, selective pressure has optimized repair mechanisms in somatic cells only to allow transmitting genetic information to the next generation, not to maximize sequence integrity long beyond the reproductive age. Recent studies have confirmed that somatic mutations, due to errors during genome repair and replication, accumulate in tissues and organs of humans and model organisms.

Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing.

Somatic mutations accumulate in multiple organs and tissues during aging and are a known cause of cancer. Cellular senescence is a possible cause of functional decline in aging, yet also acts as an anticancer mechanism . Here, we compared somatic mutation burden between early passage and deeply senescent human fibroblasts using single-cell whole-genome sequencing.

Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages.

Transcriptional profiling reveals ataxia telangiectasia mutated pathways regulate joint copper and arsenic toxicity for hepatic metalloplasia and anti-cancer therapies.

Aims: Exposures to toxic metals, including arsenic (As), pose health risks but joint effects of physiologically needed metals, e.g., copper (Cu), are ill-defined for regulated metal-dependent cell proliferation (or metalloplasia).

Single-cell analysis of somatic mutations in human bronchial epithelial cells in relation to aging and smoking.

Although lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here, we applied single-cell whole-genome sequencing of proximal bronchial basal cells from 33 participants aged between 11 and 86 years with smoking histories varying from never-smoking to 116 pack-years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers.

Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing.

Postzygotic somatic mutations have been found associated with human disease, including diseases other than cancer. Most information on somatic mutations has come from studying clonally amplified mutant cells, based on a growth advantage or genetic drift. However, almost all somatic mutations are unique for each cell, and the quantitative analysis of these low-abundance mutations in normal tissues remains a major challenge in biology.

Single-cell analysis of somatic mutation burden in mammary epithelial cells of pathogenic BRCA1/2 mutation carriers.

Inherited germline mutations in the breast cancer gene 1 (BRCA1) or BRCA2 genes (herein BRCA1/2) greatly increase the risk of breast and ovarian cancer, presumably by elevating somatic mutational errors as a consequence of deficient DNA repair. However, this has never been directly demonstrated by a comprehensive analysis of the somatic mutational landscape of primary, noncancer, mammary epithelial cells of women diagnosed with pathogenic BRCA1/2 germline mutations. Here, we used an accurate, single-cell whole-genome sequencing approach to first show that telomerized primary mammary epithelial cells heterozygous for a highly penetrant BRCA1 variant displayed a robustly elevated mutation frequency as compared with their isogenic control cells.

Maintenance of genome sequence integrity in long- and short-lived rodent species.

DNA mutations in somatic cells have been implicated in the causation of aging, with longer-lived species having a higher capacity to maintain genome sequence integrity than shorter-lived species. In an attempt to directly test this hypothesis, we used single-cell whole-genome sequencing to analyze spontaneous and bleomycin-induced somatic mutations in lung fibroblasts of four rodent species with distinct maximum life spans, including mouse, guinea pig, blind mole-rat, and naked mole-rat, as well as humans. As predicted, the mutagen-induced mutation frequencies inversely correlated with species-specific maximum life span, with the greatest difference observed between the mouse and all other species.

SomaMutDB: a database of somatic mutations in normal human tissues.

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues.

Age-related telomere attrition causes aberrant gene expression in sub-telomeric regions.

Telomere attrition has been proposed as a biomarker and causal factor in aging. In addition to causing cellular senescence and apoptosis, telomere shortening has been found to affect gene expression in subtelomeric regions. Here, we analyzed the distribution of age-related differentially expressed genes from the GTEx RNA sequencing database of 54 tissue types from 979 human subjects and found significantly more upregulated than downregulated genes in subtelomeric regions as compared to the genome-wide average.

FOXO3a acts to suppress DNA double-strand break-induced mutations.

Genomic instability is one of the hallmarks of aging, and both DNA damage and mutations have been found to accumulate with age in different species. Certain gene families, such as sirtuins and the FoxO family of transcription factors, have been shown to play a role in lifespan extension. However, the mechanism(s) underlying the increased longevity associated with these genes remains largely unknown and may involve the regulation of responses to cellular stressors, such as DNA damage.

Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan.

Accumulation of mutations in somatic cells has been implicated as a cause of aging since the 1950s. However, attempts to establish a causal relationship between somatic mutations and aging have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan using a highly accurate single-cell whole-genome sequencing method.

Genes and Pathways Promoting Long-Term Liver Repopulation by hYAP-ERT2 Transduced Hepatocytes and Treatment of Jaundice in Gunn Rats.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment.

Targeted sequencing to discover germline variants in the BRCA1 and BRCA2 genes in a Russian population and their association with breast cancer risk.

BRCA1 and BRCA2 are tumor suppressor genes involved in the repair of DNA damage and transcriptional regulation of the cell cycle. Alterations in BRCA1/2 lead to production of functionally defective proteins that impair DNA repair. Certain mutant variants of BRCA1/2 are strongly associated with increased risk of breast and ovarian cancers, with emerging data on association with other types of cancer.

Bleomycin-induced genome structural variations in normal, non-tumor cells.

Many anticancer drugs are genotoxic agents inducing DNA breaks in actively proliferating cancer cells. However, these same drugs also induce mutations, mostly genome structural variations (GSVs). The detection of GSVs in normal cells and tissues is a major challenge due to the very low abundance of these mutations, which are essentially only detectable in clonal outgrowths, such as tumors.

Accurate identification of single-nucleotide variants in whole-genome-amplified single cells.

Mutation analysis in single-cell genomes is prone to artifacts associated with cell lysis and whole-genome amplification. Here we addressed these issues by developing single-cell multiple displacement amplification (SCMDA) and a general-purpose single-cell-variant caller, SCcaller (https://github.com/biosinodx/SCcaller/).

Quantitative detection of low-abundance somatic structural variants in normal cells by high-throughput sequencing.

The detection and quantification of low-abundance somatic DNA mutations by high-throughput sequencing is challenging because of the difficulty of distinguishing errors from true mutations. There are several approaches available for analyzing somatic point mutations and small insertions or deletions, but an accurate genome-wide assessment of somatic structural variants (somSVs) in bulk DNA is still not possible. Here we present Structural Variant Search (SVS), a method to accurately detect rare somSVs by low-coverage sequencing.

High-throughput sequencing in mutation detection: A new generation of genotoxicity tests?

The advent of next generation sequencing (NGS) technology has provided the means to directly analyze the genetic material in primary cells or tissues of any species in a high throughput manner for mutagenic effects of potential genotoxic agents. In principle, direct, genome-wide sequencing of human primary cells and/or tissue biopsies would open up opportunities to identify individuals possibly exposed to mutagenic agents, thereby replacing current risk assessment procedures based on surrogate markers and extrapolations from animal studies. NGS-based tests can also precisely characterize the mutation spectra induced by genotoxic agents, improving our knowledge of their mechanism of action.

Controlled induction of DNA double-strand breaks in the mouse liver induces features of tissue ageing.

DNA damage has been implicated in ageing, but direct evidence for a causal relationship is lacking, owing to the difficulty of inducing defined DNA lesions in cells and tissues without simultaneously damaging other biomolecules and cellular structures. Here we directly test whether highly toxic DNA double-strand breaks (DSBs) alone can drive an ageing phenotype using an adenovirus-based system based on tetracycline-controlled expression of the SacI restriction enzyme. We deliver the adenovirus to mice and compare molecular and cellular end points in the liver with normally aged animals.