scholarly journals Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

2021 ◽  
Author(s):  
Yannik Bollen ◽  
Ellen Stelloo ◽  
Petra van Leenen ◽  
Myrna van den Bos ◽  
Bas Ponsioen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Single Cell ◽  
Temporal Dynamics ◽  
De Novo ◽  
Tumor Evolution ◽  
Genome Wide ◽  
Aneuploid Tumor ◽  
Evolutionary Paths ◽  
Tumor Biopsies ◽  
Punctuated Evolution

AbstractCentral to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

scholarly journals Topologically Dependent Abundance of Spontaneous DNA Damage in Single Human Cells

2019 ◽  
Author(s):  
Qiangyuan Zhu ◽  
Yichi Niu ◽  
Michael Gundry ◽  
Kuanwei Sheng ◽  
Muchun Niu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Genome Stability ◽  
De Novo ◽  
Single Cells ◽  
Human Cells ◽  
Single Nucleotide Variants ◽  
Genome Wide ◽  
First Time

AbstractIn the studies of single-cell genomics, the large endeavor has been focused on the detection of the permanent changes in the genome. On the other hand, spontaneous DNA damage frequently occurs and results in transient single-stranded changes to the genome until they are repaired. So far, successful profiling of these dynamic changes has not been demonstrated by single-cell whole-genome amplification methods. Here we reported a novel single-cell WGA method: Linearly Produced Semiamplicon based Split Amplification Reaction (LPSSAR), which allows, for the first time, the genome-wide detection of the DNA damage associated single nucleotide variants (dSNVs) in single human cells. The sequence-based detection of dSNVs allows the direct characterization of the major damage signature that occurred in human cells. In the analysis of the abundance of dSNVs along the genome, we observed two modules of dSNV abundance, instead of a homogeneous abundance of dSNVs. Interestingly, we found that the two modules are associated with the A/B topological compartments of the genome. This result suggests that the genome topology directly influences genome stability. Furthermore, with the detection of a large number of dSNVs in single cells, we showed that only under a stringent filtering condition, can we distinguish the de novo mutations from the dSNVs and achieve a reliable estimation of the total level of de novo mutations in a single cell.

Single-cell multiomics sequencing and analyses of human colorectal cancer

Science ◽  
2018 ◽  
Vol 362 (6418) ◽  
pp. 1060-1063 ◽  
Author(s):  
Shuhui Bian ◽  
Yu Hou ◽  
Xin Zhou ◽  
Xianlong Li ◽  
Jun Yong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Single Cell ◽  
Cancer Cells ◽  
Distant Metastases ◽  
Dna Demethylation ◽  
Human Colorectal Cancer ◽  
Genetic Lineages ◽  
Genome Wide ◽  
Gene Expression Dysregulation ◽  
Long Interspersed Nuclear Element

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells’ DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

scholarly journals Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm

Cell ◽  
2012 ◽  
Vol 150 (2) ◽  
pp. 402-412 ◽  
Author(s):  
Jianbin Wang ◽  
H. Christina Fan ◽  
Barry Behr ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
De Novo ◽  
Human Sperm ◽  
De Novo Mutation ◽  
Mutation Rates ◽  
Cell Analysis ◽  
Recombination Activity ◽  
Genome Wide

scholarly journals Identification of de novo mutations in the Chinese ASD cohort via whole-exome sequencing unveils brain regions implicated in autism

2021 ◽  
Author(s):  
Bo Yuan ◽  
Mengdi Wang ◽  
Xinran Wu ◽  
Peipei Cheng ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Human Brain ◽  
Single Cell ◽  
De Novo ◽  
Brain Regions ◽  
Imaging Data ◽  
Genetic Studies ◽  
Single Cell Sequencing ◽  
Genome Wide ◽  
Whole Exome ◽  
Brain Imaging Data

Abstract Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder characterized by deficits in social interactions and repetitive behaviors. Although hundreds of ASD risk genes, implicated in synaptic formation and transcriptional regulation, have been identified through human genetic studies, the East Asian ASD cohorts is still under-represented in the genome-wide genetic studies. Here we performed whole-exome sequencing on 369 ASD trios including probands and unaffected parents of Chinese origin. Using a joint-calling analytical pipeline based on GATK toolkits, we identified numerous de novo mutations including 55 high-impact variants and 165 moderate-impact variants, as well as de novo copy number variations containing known ASD-related genes. Importantly, combining with single-cell sequencing data from the developing human brain, we found that expression of genes with de novo mutations were specifically enriched in pre-, post-central gyrus (PRC, PC) and banks of superior temporal (BST) regions in the human brain. By further analyzing the brain imaging data with ASD and health controls, we found that the gray volume of the right BST in ASD patients significantly decreased comparing to health controls, suggesting the potential structural deficits associated with ASD. Finally, we found that there was decrease in the seed-based functional connectivity (FC) between BST/PC/PRC and sensory areas, insula, as well as frontal lobes in ASD patients. This work indicated that the combinatorial analysis with genome-wide screening, single-cell sequencing and brain imaging data would reveal brain regions contributing to etiology of ASD.

scholarly journals Single-Cell Sequencing Technology in Oncology: Applications for Clinical Therapies and Research

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Baixin Ye ◽  
Qingping Gao ◽  
Zhi Zeng ◽  
Creed M. Stary ◽  
Zhihong Jian ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Descriptive Analysis ◽  
Genetic Material ◽  
Cellular Heterogeneity ◽  
Cancer Therapies ◽  
Single Cell Sequencing ◽  
Targeted Analysis ◽  
Genome Wide ◽  
Tumor Biopsies

Cellular heterogeneity is a fundamental characteristic of many cancers. A lack of cellular homogeneity contributes to difficulty in designing targeted oncological therapies. Therefore, the development of novel methods to determine and characterize oncologic cellular heterogeneity is a critical next step in the development of novel cancer therapies. Single-cell sequencing (SCS) technology has been recently employed for analyzing the genetic polymorphisms of individual cells at the genome-wide level. SCS requires (1) precise isolation of the single cell of interest; (2) isolation and amplification of genetic material; and (3) descriptive analysis of genomic, transcriptomic, and epigenomic data. In addition to targeted analysis of single cells isolated from tumor biopsies, SCS technology may be applied to circulating tumor cells, which may aid in predicting tumor progression and metastasis. In this paper, we provide an overview of SCS technology and review the current literature on the potential application of SCS to clinical oncology and research.

scholarly journals Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

2018 ◽  
Author(s):  
Stephanie Lauer ◽  
Grace Avecilla ◽  
Pieter Spealman ◽  
Gunjan Sethia ◽  
Nathan Brandt ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Tandem Repeats ◽  
Evolutionary Dynamics ◽  
Temporal Dynamics ◽  
De Novo ◽  
Common Mechanism ◽  
Second Phase ◽  
Evolutionary Potential ◽  
Ideal System

AbstractCopy number variants (CNVs) are a pervasive, but understudied source of genetic variation and evolutionary potential. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics ofde novoCNVs. Here, we developed a fluorescent reporter to monitor gene amplifications and deletions at a specific locus with single-cell resolution. Using a CNV reporter in nitrogen-limited chemostats, we find thatGAP1CNVs are repeatedly generated and selected during the early stages of adaptive evolution resulting in predictable dynamics of CNV selection. However, subsequent diversification of populations defines a second phase of evolutionary dynamics that cannot be predicted. Using whole genome sequencing, we identified a variety ofGAP1CNVs that vary in size and copy number. DespiteGAP1’s proximity to tandem repeats that facilitate intrachromosomal recombination, we find that non-allelic homologous recombination (NAHR) between flanking tandem repeats occurs infrequently. Rather, breakpoint characterization revealed that for at least 50% ofGAP1CNVs, origin-dependent inverted-repeat amplification (ODIRA), a DNA replication mediated process, is the likely mechanism. We also find evidence that ODIRA generatesDUR3CNVs, indicating that it may be a common mechanism of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 103-104independent CNV-containing lineages initially compete within populations, which results in extreme clonal interference. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into the underlying dynamics of CNVs in evolution.

scholarly journals Proof-of-concept pilot study on comprehensive spatiotemporal intra-patient heterogeneity for colorectal cancer with liver metastasis

2021 ◽  
Author(s):  
Ioannis Dimitrios Kyrochristos ◽  
Georgios K. Glantzounis ◽  
Anna Goussia ◽  
Alexia Eliades ◽  
Achilleas Achilleos ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
De Novo ◽  
Tumor Resection ◽  
Tumor Evolution ◽  
Precision Oncology ◽  
Proof Of Concept ◽  
De Novo Mutations ◽  
Liquid Biopsies ◽  
Patient Heterogeneity

Purpose: The mechanisms underlying high drug resistance and relapse rates after multi-modal treatment in patients with colorectal cancer (CRC) and liver metastasis (LM) remain poorly understood. We evaluate the potential translational implications of intra-patient heterogeneity (IPH) comprising primary and matched metastatic intratumor heterogeneity (ITH) coupled with circulating tumor DNA (ctDNA) variability. Patients and methods: According to our IPH-based protocol, 18 eligible patients with CRC-LM, who underwent complete tumor resection after neo-adjuvant treatment, with a total of 122 multi-regional tumor and perioperative liquid biopsies were analyzed via next-generation sequencing (NGS) of a custom 77-gene panel. The primary endpoints were the extent of IPH and the frequency of actionable mutations. Results: The proportion of patients with ITH were 53% and 56% in primary CRC and LM respectively, while 35% of patients harbored de novo mutations in LM indicating spatiotemporal tumor evolution and the necessity of multiregional analysis. Among the 56% of patients with alterations in liquid biopsies, de novo mutations in cfDNA were identified in 25% of patients, which were undetectable in both CRC and LM. All 17 patients with driver alterations harbored actionable mutations, with an average of 3.2 oncogenic events per patient, for molecularly targeted drugs either approved or under evaluation in ongoing clinical trials or in pre-clinical studies. Conclusions: Our proof-of-concept prospective study provides initial evidence and warrants the conduction of precision oncology trials to test the potential clinical utility of IPH-driven matched therapy.

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