scholarly journals A high-fidelity method for genomic sequencing of single somatic cells reveals a very high mutational burden

2017 ◽  
Vol 242 (13) ◽  
pp. 1318-1324 ◽  
Author(s):  
Jan Vijg ◽  
Xiao Dong ◽  
Lei Zhang
Keyword(s):  
Single Cell ◽  
Somatic Mutations ◽  
Somatic Cells ◽  
Whole Genome ◽  
Tissue Samples ◽  
Amplification System ◽  
Functional Consequences ◽  
Postzygotic Mutations

Postzygotic mutations in somatic cells lead to genome mosaicism and can be the cause of cancer, possibly other human diseases and aging. Somatic mutations are difficult to detect in bulk tissue samples. Here, we review the available assays for measuring somatic mutations, with a focus on recent single-cell, whole genome sequencing methods. Impact statement Somatic mutations cause cancer, possibly other diseases and aging. Yet, very little is known about the frequency of such mutations in vivo, their distribution across the genome, and their possible functional consequences other than cancer. Even in cancer, we do not know the heterogeneity of mutations within a tumor and if seemingly normal cells in its surroundings already have elevated mutation frequencies. Here, we review a new, whole genome amplification system that allows accurate quantification and characterization of single-cell mutational landscapes in human cells and tissues in relation to disease.

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

scholarly journals Molecular Characterization of HOXA2 and HOXA3 Binding Properties

2021 ◽  
Vol 9 (4) ◽  
pp. 55
Author(s):  
Joshua Mallen ◽  
Manisha Kalsan ◽  
Peyman Zarrineh ◽  
Laure Bridoux ◽  
Shandar Ahmad ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Molecular Characterization ◽  
Sequence Motif ◽  
Body Parts ◽  
Binding Affinities ◽  
Binding Properties ◽  
Functional Consequences

The highly conserved HOX homeodomain (HD) transcription factors (TFs) establish the identity of different body parts along the antero–posterior axis of bilaterian animals. Segment diversification and the morphogenesis of different structures is achieved by generating precise patterns of HOX expression along the antero–posterior axis and by the ability of different HOX TFs to instruct unique and specific transcriptional programs. However, HOX binding properties in vitro, characterised by the recognition of similar AT-rich binding sequences, do not account for the ability of different HOX to instruct segment-specific transcriptional programs. To address this problem, we previously compared HOXA2 and HOXA3 binding in vivo. Here, we explore if sequence motif enrichments observed in vivo are explained by binding affinities in vitro. Unexpectedly, we found that the highest enriched motif in HOXA2 peaks was not recognised by HOXA2 in vitro, highlighting the importance of investigating HOX binding in its physiological context. We also report the ability of HOXA2 and HOXA3 to heterodimerise, which may have functional consequences for the HOX patterning function in vivo.

scholarly journals Characterization of in vivo somatic mutations at the hypoxanthine phosphoribosyltransferase gene of a human control population.

1993 ◽  
Vol 101 (1) ◽  
pp. 68-74 ◽  
Author(s):  
K Burkhart-Schultz ◽  
C B Thomas ◽  
C L Thompson ◽  
C L Strout ◽  
E Brinson ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Control Population ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Human Control

Functional consequences of contrasting properties of α1/α2 systems

1985 ◽  
Vol 68 (s10) ◽  
pp. 93s-97s ◽  
Author(s):  
Pieter B. M. W. M. Timmermans ◽  
Martin J. M. C. Thoolen ◽  
Adriaan De Jonge ◽  
Bob Wilffert ◽  
Pieter A. Van Zwieten
Keyword(s):  
Smooth Muscle ◽  
Experimental Evidence ◽  
Diastolic Pressure ◽  
Previous Treatment ◽  
Pithed Rat ◽  
Pithed Rats ◽  
Functional Consequences ◽  
Adrenoceptor Agonists

Ample experimental evidence supports the existence of two distinct types of vasoconstrictor α-adrenoceptors in vascular smooth muscle at postjunctional sites, showing similarities with α1 and α2-adrenoceptors. Especially in vivo, the characterization of an α2-adrenoceptor mediating an increase in diastolic pressure has been most successful. In this respect the model of the pithed rat has been employed most frequently (for reviews see [1, 2]). At present, the agents cirazoline, (−) phenylephrine, (±)-erythro-methoxamine, (−)-amidephrine, SKF89748, St587 and Sgd 101/75 fulfil the criteria for selective α1-adrenoceptor agonists; their log dose-vasopressor effect curves are virtually unaffected by previous treatment with yohimbine or rauwolscine (selective dose in pithed rats: 1 mg/kg), whereas prazosin (selective dose in pithed rats: 0.1 mg/kg) causes an appreciable parallel rightward displacement. The reverse holds true for the stimulants B-HT 920, B-HT 933, B-HT 958, UK-14304, xylazine, TL-99, M-7 and DP-6,7-ADTN, which all have been classified as preferential agonists of vascular postjunctional α-adrenoceptors.

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

2019 ◽  
Author(s):  
Lei Zhang ◽  
Xiao Dong ◽  
Moonsook Lee ◽  
Alexander Y. Maslov ◽  
Tao Wang ◽  
...  
Keyword(s):  
B Cell ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
B Lymphocytes ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Somatic Hypermutation ◽  
Whole Genome ◽  
Healthy Individuals ◽  
Human Lifespan

Introductory paragraphThe accumulation of mutations in somatic cells have been implicated as a cause of ageing since the 1950s1,2. Yet, attempts to establish a causal relationship between somatic mutations and ageing have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide detailed, genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan, from newborns to centenarians, using a recently developed, highly accurate single-cell whole-genome sequencing method3. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, located at immunoglobulin genes associated with somatic hypermutation. B cell-specific mutation signatures were observed associated with development, ageing or somatic hypermutation (SHM). The SHM signature strongly correlated with the signature found in human chronic lymphocytic leukemia and malignant B-cell lymphomas4, indicating that even in B cells of healthy individuals the potential cancer-causing events are already present. We also identified multiple mutations in sequence features relevant to cellular function, i.e., transcribed genes and gene regulatory regions. Such mutations increased significantly during ageing, but only at approximately half the rate of the genome average, indicating selection against mutations that impact B cell function. This first full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

scholarly journals Identification of Somatic Mutations From Bulk and Single-Cell Sequencing Data

2022 ◽  
Vol 2 ◽  
Author(s):  
August Yue Huang ◽  
Eunjung Alice Lee
Keyword(s):  
Single Cell ◽  
Somatic Mutations ◽  
Sequencing Data ◽  
Tissue Samples ◽  
Genome Amplification ◽  
Bioinformatic Tools ◽  
Base Calling ◽  
Human Lifespan ◽  
Dna Variants ◽  
Underlying Mechanisms

Somatic mutations are DNA variants that occur after the fertilization of zygotes and accumulate during the developmental and aging processes in the human lifespan. Somatic mutations have long been known to cause cancer, and more recently have been implicated in a variety of non-cancer diseases. The patterns of somatic mutations, or mutational signatures, also shed light on the underlying mechanisms of the mutational process. Advances in next-generation sequencing over the decades have enabled genome-wide profiling of DNA variants in a high-throughput manner; however, unlike germline mutations, somatic mutations are carried only by a subset of the cell population. Thus, sensitive bioinformatic methods are required to distinguish mutant alleles from sequencing and base calling errors in bulk tissue samples. An alternative way to study somatic mutations, especially those present in an extremely small number of cells or even in a single cell, is to sequence single-cell genomes after whole-genome amplification (WGA); however, it is critical and technically challenging to exclude numerous technical artifacts arising during error-prone and uneven genome amplification in current WGA methods. To address these challenges, multiple bioinformatic tools have been developed. In this review, we summarize the latest progress in methods for identification of somatic mutations and the challenges that remain to be addressed in the future.

scholarly journals The origins and functional effects of postzygotic mutations throughout the human lifespan

2021 ◽  
Author(s):  
Nicole Briana Rockweiler ◽  
Avinash Ramu ◽  
Liina Nagirnaja ◽  
Wing Hing Wong ◽  
Michiel J. Noordam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Phylogenetic Reconstruction ◽  
Tissue Expression ◽  
Prenatal Development ◽  
Tissue Samples ◽  
Human Lifespan ◽  
Specific Effects ◽  
Mutation Burden ◽  
Functional Consequences ◽  
Postzygotic Mutations

Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remains unclear. To study the origins and functional consequences of PZMs, we generated a multi-tissue atlas of PZMs from 948 donors using the final major release of the Genotype-Tissue Expression (GTEx) project. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, while 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we find that their type and predicted functional impact varies during prenatal development, across tissues, and the germ cell lifecycle. Remarkably, a class of prenatal mutations was predicted to be more deleterious than any other category of genetic variation investigated and under positive selection as strong as somatic mutations in cancers. In total, the data indicate that PZMs can contribute to phenotypic variation throughout the human lifespan, and, to better understand the relationship between genotype and phenotype, we must broaden the long-held assumption of one genome per individual to multiple, dynamic genomes per individual.

scholarly journals Systematic Comparison of High-Throughput Single-Cell RNAseq Methods for Immune Cell Profiling

2020 ◽  
Author(s):  
Chi-Ming Kevin Li ◽  
Tracy M Yamawaki ◽  
Daniel R Lu ◽  
Daniel C Ellwanger ◽  
Dev Bhatt ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Drop Out ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the fieldof immunology by deepening the characterization of immune heterogeneity and leading to thediscovery of new subtypes. However, single-cell methods inherently suffer from limitations in therecovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropoutevents. This issue is often compounded by limited sample availability and limited prior knowledge ofheterogeneity, which can confound data interpretation.Results: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. Weprepared 21 libraries under identical conditions of a defined mixture of two human and two murinelymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluatemethods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expressionsignatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5’v1 and 3’ v3 methods. We demonstrate that these methods have fewer drop-out events whichfacilitates the identification of differentially-expressed genes and improves the concordance of singlecellprofiles to immune bulk RNA-seq signatures.Conclusion: Overall, our characterization of immune cell mixtures provides useful metrics, which canguide selection of a high-throughput single-cell RNA-seq method for profiling more complex immunecellheterogeneity usually found in vivo.

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

2019 ◽  
Vol 116 (18) ◽  
pp. 9014-9019 ◽  
Author(s):  
Lei Zhang ◽  
Xiao Dong ◽  
Moonsook Lee ◽  
Alexander Y. Maslov ◽  
Tao Wang ◽  
...  
Keyword(s):  
B Cell ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
B Lymphocytes ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Whole Genome ◽  
Healthy Individuals ◽  
Human Lifespan ◽  
Increased Risk

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. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, were located at Ig genes associated with somatic hypermutation (SHM). B cell–specific mutation signatures associated with development, aging, or SHM were found. The SHM signature strongly correlated with the signature found in human B cell tumors, indicating that potential cancer-causing events are already present even in B cells of healthy individuals. We also identified multiple mutations in sequence features relevant to cellular function (i.e., transcribed genes and gene regulatory regions). Such mutations increased significantly during aging, but only at approximately one-half the rate of the genome average, indicating selection against mutations that impact B cell function. This full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

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