ABSTRACT
How many times does a typical hematopoietic stem cell (HSC) divide to maintain a daily production of over 1011 blood cells over a human lifetime? It has been predicted that relatively few, slowly dividing HSCs occupy the top of the hematopoietic hierarchy. However, tracking HSCs directly is extremely challenging due to their rarity. Here, we utilize previously published data documenting the loss of telomeric DNA repeats in granulocytes, to draw inferences about HSC division rates, the timing of major changes in those rates, as well as lifetime division totals. Our method uses segmented regression to identify the best candidate representations of the telomere length data. Our method predicts that, on average, an HSC divides 56 times over an 85-year lifespan (with lower and upper bounds of 36 and 120, respectively), with half of these divisions during the first 24 years of life.
ABSTRACT
Hundreds of loci in human genomes have alleles that are methylated differentially according to their parent of origin. These imprinted loci generally show little variation across tissues, individuals, and populations. We show that such loci can be used to distinguish the maternal and paternal homologs for all human autosomes without the need for the parental DNA. We integrate methylation-detecting nanopore sequencing with the long-range phase information in Strand-seq data to determine the parent of origin of chromosome-length haplotypes for both DNA sequence and DNA methylation in five trios with diverse genetic backgrounds. The parent of origin was correctly inferred for all autosomes with an average mismatch error rate of 0.31% for SNVs and 1.89% for insertions or deletions (indels). Because our method can determine whether an inherited disease allele originated from the mother or the father, we predict that it will improve the diagnosis and management of many genetic diseases.
ABSTRACT
Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.
Subject(s)
Pluripotent Stem Cells , Telomerase , Humans , Telomere Homeostasis/genetics , Telomere/genetics , Cell Differentiation/genetics , Telomerase/genetics , X-linked Nuclear Protein/geneticsABSTRACT
Dense local haplotypes can now readily be extracted from long-read or droplet-based sequence data. However, these methods struggle to combine subchromosomal haplotype blocks into global chromosome-length haplotypes. Strand-seq is a single cell sequencing technique that uses read orientation to capture sparse global phase information by sequencing only one of two DNA strands for each parental homolog. In combination with dense local haplotypes from other technologies, Strand-seq data can be used to obtain complete chromosome-length phase information. In this chapter, we run the R package StrandPhaseR to phase SNVs using publicly available sequence data for sample HG005 of the Genome in a Bottle project.
Subject(s)
Chromosomes , Genome , Haplotypes , Sequence Analysis, DNA/methods , High-Throughput Nucleotide Sequencing/methods , Polymorphism, Single Nucleotide , AlgorithmsABSTRACT
Telomeres and telomerase play a crucial role in human aging and cancer. Three "drivers" of human aging can be identified. The developmental program encoded in DNA is the primary determinant of lifespan. Faithful execution of the developmental program requires stability of the (epi-)genome which is challenged throughout life by damage to DNA as well as epigenetic 'scars' from error-free DNA repair and stochastic errors made during the establishment and maintenance of the "epigenome". Over time (epi-)mutations accumulate, compromising cellular function and causing (pre-)malignant alterations. Damage to the genome and epigenome can be considered the second "driver" of aging. A third driver of the aging process, important to suppress tumors in long-lived animals, is caused by progressive loss of telomeric DNA. Telomere erosion protects against cancer early in life but limits cell renewal late in life, in agreement with the Antagonistic Pleiotropy theory on the evolutionary origin of aging. Malignant tumors arise when mutations and/or epimutations in cells (clock 2) corrupt the developmental program (clock 1) as well as tumor suppression by telomere erosion (clock 3). In cancer cells clock 3 is typically inactivated by loss of p53 as well as increased expression of telomerase. Taken together, aging in humans can be described by the ticking of three clocks: the clock that directs development, the accumulation of (epi-)mutations over time and the telomere clock that limits the number of cell divisions in normal stem and immune cells.
Subject(s)
Neoplasms , Telomerase , Animals , Humans , Telomerase/genetics , Telomerase/metabolism , Telomere/genetics , Telomere/metabolism , Neoplasms/genetics , Aging/geneticsABSTRACT
Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors , Sister Chromatid Exchange , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Chromosome Fragile Sites , Homologous Recombination/genetics , DNAABSTRACT
Polymorphic inversions are ubiquitous in humans and they have been linked to both adaptation and disease. Following their discovery in Drosophila more than a century ago, inversions have proved to be more elusive than other structural variants. A wide variety of methods for the detection and genotyping of inversions have recently been developed: multiple techniques based on selective amplification by PCR, short- and long-read sequencing approaches, principal component analysis of small variant haplotypes, template strand sequencing, optical mapping, and various genome assembly methods. Many methods apply complex wet lab protocols or increasingly refined bioinformatic analyses. This review is an attempt to provide a practical summary and comparison of the methods that are in current use, with a focus on metrics such as the maximum size of segmental duplications at inversion breakpoints that each method can tolerate, the size range of inversions that they recover, their throughput, and whether the locations of putative inversions must be known beforehand.
Subject(s)
Chromosome Inversion , Drosophila , Animals , Chromosome Inversion/genetics , Drosophila/genetics , Genotype , Haplotypes , Humans , Principal Component AnalysisABSTRACT
Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles. By investigating the impact of monosomies, trisomies and segmental aneuploidies on chromosomal instability we further deciphered the complex relationship between aneuploidy and CIN.
Subject(s)
Aneuploidy , Trisomy , Chromosomal Instability , Genetic Testing , Humans , Monosomy , Trisomy/geneticsABSTRACT
Telomerase levels in most human cells are insufficient to prevent loss of telomeric DNA with each replication cycle. The resulting "Hayflick" limit may have allowed lifespan to increase by suppressing the development of tumors early in life be it at the expense of compromised cellular responses late in life. At any given age, the average telomere length in leukocytes shows considerably variation between individuals with females having, on average, longer telomeres than males. Sex differences in average telomere length are already present at birth and correspond to reported differences in the average life expectancy between the sexes. Levels of telomerase RNA and dyskerin, encoded by DKC1, are known to limit telomerase activity in embryonic stem cells. X-linked DKC1 is expressed from both alleles in female embryo cells and higher levels of dyskerin and telomerase could elongate telomeres prior to embryo implantation. The hypothesis that embryonic telomerase levels set the stage for the sex differences in telomere length and lifespan deserves further study.
Subject(s)
Dyskeratosis Congenita , Telomerase , Cell Cycle Proteins/metabolism , Dyskeratosis Congenita/genetics , Dyskeratosis Congenita/pathology , Female , Humans , Infant, Newborn , Longevity/genetics , Male , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Sex Characteristics , Telomerase/genetics , Telomerase/metabolism , Telomere/metabolismABSTRACT
Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput.
Subject(s)
Genomics , Genomics/methods , HaplotypesABSTRACT
Mammalian genomes encode over a hundred different helicases, many of which are implicated in the repair of DNA lesions by acting on DNA structures arising during DNA replication, recombination or transcription. Defining the in vivo substrates of such DNA helicases is a major challenge given the large number of helicases in the genome, the breadth of potential substrates in the genome and the degree of genetic pleiotropy among DNA helicases in resolving diverse substrates. Helicases such as WRN, BLM and RECQL5 are implicated in the resolution of error-free recombination events known as sister chromatid exchange events (SCEs). Single cell Strand-seq can be used to map the genomic location of individual SCEs at a resolution that exceeds that of classical cytogenetic techniques by several orders of magnitude. By mapping the genomic locations of SCEs in the absence of different helicases, it should in principle be possible to infer the substrate specificity of specific helicases. Here we describe how the genome can be interrogated for such DNA repair events using single-cell template strand sequencing (Strand-seq) and bioinformatic tools. SCEs and copy-number alterations were mapped to genomic locations at kilobase resolution in haploid KBM7 cells. Strategies, possibilities, and limitations of Strand-seq to study helicase function are illustrated using these cells before and after CRISPR/Cas9 knock out of WRN, BLM and/or RECQL5.
Subject(s)
DNA Replication , Sister Chromatid Exchange , Animals , DNA/chemistry , DNA/genetics , DNA Repair/genetics , DNA Replication/genetics , Genome , Mammals , Sister Chromatid Exchange/geneticsABSTRACT
The role of telomeres in human health and disease is yet to be fully understood. The limitations of mouse models for the study of human telomere biology and difficulties in accurately measuring the length of telomere repeats in chromosomes and cells have diverted attention from many important and relevant observations. The goal of this perspective is to summarize some of these observations and to discuss the antagonistic role of telomere loss in aging and cancer in the context of developmental biology, cell turnover, and evolution. It is proposed that both damage to DNA and replicative loss of telomeric DNA contribute to aging in humans, with the differences in leukocyte telomere length between humans being linked to the risk of developing specific diseases. These ideas are captured in the Telomere Erosion in Disposable Soma theory of aging proposed herein.
Subject(s)
Aging , Neoplasms/genetics , Telomere Shortening , Telomere/genetics , Animals , DNA/genetics , DNA/metabolism , DNA Damage , Disease Models, Animal , Humans , Mutation , Neoplasms/metabolism , Telomerase/genetics , Telomerase/metabolism , Telomere/metabolismABSTRACT
Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.
Subject(s)
Chromatin/metabolism , DNA Replication/physiology , Hydroxyurea/pharmacology , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Chromatin/chemistry , Gene Expression Regulation/drug effects , Humans , Stress, Physiological , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Single cell Strand-seq is a unique tool for the discovery and phasing of genomic inversions. Conventional methods to discover inversions with Strand-seq data are blind to known inversion locations, limiting their statistical power for the detection of inversions smaller than 10 Kb. Moreover, the methods rely on manual inspection to separate false and true positives. RESULTS: Here we describe "InvertypeR", a method based on a Bayesian binomial model that genotypes inversions using fixed genomic coordinates. We validated InvertypeR by re-genotyping inversions reported for three trios by the Human Genome Structural Variation Consortium. Although 6.3% of the family inversion genotypes in the original study showed Mendelian discordance, this was reduced to 0.5% using InvertypeR. By applying InvertypeR to published inversion coordinates and predicted inversion hotspots (n = 3701), as well as coordinates from conventional inversion discovery, we furthermore genotyped 66 inversions not previously reported for the three trios. CONCLUSIONS: InvertypeR discovers, genotypes, and phases inversions without relying on manual inspection. For greater accessibility, results are presented as phased chromosome ideograms with inversions linked to Strand-seq data in the genome browser. InvertypeR increases the power of Strand-seq for studies on the role of inversions in phenotypic variation, genome instability, and human disease.
Subject(s)
Chromosome Inversion , Genome, Human , Bayes Theorem , Genotype , Haplotypes , HumansABSTRACT
Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Single-Cell Analysis/methods , Whole Genome Sequencing/methods , Algorithms , Alleles , Animals , Base Sequence , Chromosome Mapping/methods , Chromosomes , Genomics/methods , Humans , Sequence Analysis, DNA/methods , SoftwareABSTRACT
Human genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing1,2 with continuous long-read or high-fidelity3 sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.
Subject(s)
Genome, Human , High-Throughput Nucleotide Sequencing/methods , Parents , Sequence Analysis, DNA/methods , Single-Cell Analysis/methods , Algorithms , Haplotypes , Humans , Puerto Rico/ethnologyABSTRACT
Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by γ-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.
Subject(s)
Embryonic Development , Spermatozoa , Animals , Cattle , DNA Damage , Embryonic Development/genetics , Female , Genomic Instability , Humans , Male , Mosaicism , PregnancyABSTRACT
Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (≈2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch® and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 × 108 leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products-16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10-20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p < 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2-6, CellSearch median CTC/mL = 0.9, IQR = 0-1.8, p < 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45-, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.
ABSTRACT
PURPOSE: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. EXPERIMENTAL DESIGN: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. RESULTS: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. CONCLUSIONS: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.
Subject(s)
Biomarkers, Tumor/blood , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/surgery , Neoplastic Cells, Circulating/pathology , Pulmonary Veins/pathology , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/blood , Epithelial Cells/pathology , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/pathology , Lung Neoplasms/surgery , Male , Middle Aged , Neoplastic Cells, Circulating/metabolism , Prospective Studies , Pulmonary Veins/metabolismABSTRACT
MOTIVATION: Strand-seq is a specialized single-cell DNA sequencing technique centered around the directionality of single-stranded DNA. Computational tools for Strand-seq analyses must capture the strand-specific information embedded in these data. RESULTS: Here we introduce breakpointR, an R/Bioconductor package specifically tailored to process and interpret single-cell strand-specific sequencing data obtained from Strand-seq. We developed breakpointR to detect local changes in strand directionality of aligned Strand-seq data, to enable fine-mapping of sister chromatid exchanges, germline inversion and to support global haplotype assembly. Given the broad spectrum of Strand-seq applications we expect breakpointR to be an important addition to currently available tools and extend the accessibility of this novel sequencing technique. AVAILABILITY AND IMPLEMENTATION: R/Bioconductor package https://bioconductor.org/packages/breakpointR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.