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.

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.

Expression of retrotransposons contributes to aging in Drosophila.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis.

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.

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.

A workflow for simultaneous DNA copy number and methylome analysis of inner cell mass and trophectoderm cells from human blastocysts.

Objective: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development.

Design: Experimental.

Setting: Academic medical center.

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.

A direct comparison of interphase FISH versus low-coverage single cell sequencing to detect aneuploidy reveals respective strengths and weaknesses.

Aneuploidy has been reported to occur at remarkably high levels in normal somatic tissues using Fluorescence In Situ Hybridization (FISH). Recently, these reports were contradicted by single-cell low-coverage whole genome sequencing (scL-WGS) analyses, which showed aneuploidy frequencies at least an order of magnitude lower. To explain these seemingly contradictory findings, we used both techniques to analyze artificially generated mock aneuploid cells and cells with natural random aneuploidy.

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.

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.

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells.

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.

The progeroid phenotype of Ku80 deficiency is dominant over DNA-PKCS deficiency.

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80-/- mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs-/- mice. However, these observations are based on independent studies with varying genetic backgrounds.

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair.

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70(-/-) cells and ku80(-/-) cells also appeared to have a defect in base excision repair (BER).

Myc-dependent genome instability and lifespan in Drosophila.

The Myc family of transcription factors are key regulators of cell growth and proliferation that are dysregulated in a large number of human cancers. When overexpressed, Myc family proteins also cause genomic instability, a hallmark of both transformed and aging cells. Using an in vivo lacZ mutation reporter, we show that overexpression of Myc in Drosophila increases the frequency of large genome rearrangements associated with erroneous repair of DNA double-strand breaks (DSBs).

Loss of the bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila.

Background: The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood.

Universal parameters for carbon nanotube network-based sensors: can nanotube sensors be reproducible?

Carbon nanotube (CNT) network-based sensors have been often considered unsuitable for practical applications due to their unpredictable characteristics. Herein, we report the study of universal parameters which can be used to characterize CNT network-based sensors and make their response predictable. A theoretical model is proposed to explain these parameters, and sensing experiments for mercury (Hg(2+)) and ammonium (NH(4)(+)) ions using CNT network-based sensors were performed to confirm the validity of our model.