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Current Protocols in Human Genetics

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https://www.readbyqxmd.com/read/29989334/assessing-skewed-x-chromosome-inactivation
#1
Thomas Liehr, Monika Ziegler, Sharon Löhmer, Anja Weise
We describe a simple and straightforward method for detection and characterization of X-chromosome inactivation in females and/or individuals with more than one X chromosome. The X-chromosome inactivation pattern is visualized on a single-cell level using 5-ethynyl-2-deoxyuridine (EdU) instead of the previously widely applied 5-bromo-2'-deoxyuridine (BUdR). The fluorochrome-labeled nucleoside analog EdU is incorporated into late-replication chromosomal regions of living blood cells in vitro; thus, it can also be used to specifically highlight the inactive X chromosome within a cytogenetic preparation...
July 10, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29979828/reporting-of-clinical-genome-sequencing-results
#2
Cui Song, Hatice Duzkale, Jun Shen
High-throughput sequencing and high-performance computing technologies have become powerful tools in clinical genetic diagnosis of hereditary disorders and genetic screening of healthy individuals to provide information for the diagnosis, treatment, and prevention of diseases or impairment and assessment of health. For patients with undiagnosed disorders, including many rare disorders, the whole-genome sequencing (WGS) test may end the diagnostic odyssey, ultimately guiding clinical care for them and their families...
July 6, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29979824/efficient-differentiation-of-human-pluripotent-stem-cells-to-endothelial-cells
#3
Mingxia Gu
Endothelial cells (ECs) line the interior surface of blood and lymphatic vessels, and play a key role in a variety of physiological or pathological processes such as thrombosis, inflammation, or vascular wall remodeling. Human-induced pluripotent stem cell (iPSCs)-derived ECs provide a new opportunity for vascular regeneration and serve as a model to study the mechanism and to screen for novel therapies. We use developmental cues in a monolayer differentiation approach to efficiently generate mesoderm cells from iPSCs via small-molecule activation of WNT signaling in chemically defined medium for 4 days, and subsequent EC specification using vascular endothelial growth factor and fibroblast growth factor for another 4 days...
July 6, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29979818/capture-hi-c-library-generation-and-analysis-to-detect-chromatin-interactions
#4
Giulia Orlando, Ben Kinnersley, Richard S Houlston
Chromosome conformation capture (3C), coupled with next-generation sequencing (Hi-C), provides a means for deciphering not only the principles underlying genome folding and architecture, but more broadly, the role 3D chromatin structure plays in gene regulation and the replication and repair of DNA. The recently implemented modification, in situ Hi-C, maintains nuclear integrity during digestion and ligation steps, reducing random ligation of Hi-C fragments. Although Hi-C allows for genome-wide characterization of chromatin contacts, it requires high-depth sequencing to discover significant contacts...
July 6, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29975818/using-xcavator-and-excavator2-to-identify-cnvs-from-wgs-wes-and-ts-data
#5
Romina D'Aurizio, Roberto Semeraro, Alberto Magi
Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing experiments (whole-genome, whole-exome, and targeted) in various disease contexts and population genetic studies...
July 5, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29953168/preparation-culture-and-analysis-of-amniotic-fluid-samples
#6
Patricia Minehart Miron
Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid may be used directly for biochemical analyses, fluorescence in situ hybridization (FISH), and isolation of DNA for molecular studies, including chromosomal microarray analysis (CMA). The fluid is typically cultured as a source of metaphase cells for chromosome analysis and to provide additional material for biochemical and DNA-based testing. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for metaphase chromosome analysis...
June 28, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/30040223/pedigree-selection-and-information-content
#7
Badri N Vardarajan, Gary W Beecham, Jonathan L Haines
In this article, we discuss strategies for selection of families and family members for genetic studies. We will evaluate strategies to sample large families with multiply affected members, sibships, and nuclear families. In addition, we have added a section to discuss sub-sampling within pedigrees for large sequencing studies, particularly when genome-wide SNP chips are available on all members of a pedigree. The type of family sampled for a study will determine the statistical analyses and power of discovery of genetic findings...
April 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/30040209/droplet-digital-pcr-with-evagreen-assay-confirmational-analysis-of-structural-variants
#8
Angela C Tai, Michael Parfenov, Joshua M Gorham
DNA structural variants can be analyzed by droplet digital PCR (ddPCR), a water-oil microfluidics and fluorescence technology to quantify target nucleic acids with extreme precision and sensitivity. Traditional ddPCR uses expensive fluorescent oligonucleotide probes that require extensive optimization. Here we describe a variation of ddPCR using a DNA-binding dye (EvaGreen), whose properties allow target products to be effectively quantified at a significantly lower cost. © 2018 by John Wiley & Sons, Inc...
April 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/30040203/plink-key-functions-for-data-analysis
#9
Susan H Slifer
Genetic data analysis of large numbers of single nucleotide variants (SNVs), including genome-wide association studies (GWAS), exome chips, and whole exome (WES) or whole-genome (WGS) sequencing data, requires well defined processing steps. As a result, several freely available analytic toolkits have been developed to streamline these processes. Among these, PLINK is the most comprehensive in terms of its quality control and analytic modules, although its focus remains on SNVs. PLINK fulfills two analytic needs-aiding the process of performing quality control (QC) on large data sets and providing basic statistical tools to analyze the variants in genetic models...
April 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/30038699/computational-prediction-of-position-effects-of-human-chromosome-rearrangements
#10
Cinthya J Zepeda-Mendoza, Shreya Menon, Cynthia C Morton
Balanced and apparently balanced chromosome abnormalities (BCAs) have long been known to generate disease through position effects, either by altering local networks of gene regulation or positioning genes in architecturally different chromosome domains. Despite these observations, identification of distally affected genes by BCAs is oftentimes neglected, especially when predicted gene disruptions are found elsewhere in the genome. In this unit, we provide detailed instructions on how to run a computational pipeline that identifies relevant candidates of non-coding BCA position effects...
April 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/30038698/in-vivo-and-in-vitro-methods-to-identify-dna-sequence-variants-that-alter-rna-splicing
#11
Parth N Patel, Joshua M Gorham, Kaoru Ito, Christine E Seidman
Identification of sequence variants that create or eliminate splice sites has proven to be a significant challenge and represents one of many roadblocks in the clinical interpretation of rare genetic variation. Current methods of identifying splice altering sequence variants exist, however, these are limited by an imperfect understanding of splice signals and cumbersome functional assays. We have recently developed a computational tool that prioritizes putative splice-altering sequence variants, and a moderate-throughput minigene assay that confirms the variants which alter splicing...
April 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29364522/differentiation-and-contractile-analysis-of-gfp-sarcomere-reporter-hipsc-cardiomyocytes
#12
Arun Sharma, Christopher N Toepfer, Manuel Schmid, Amanda C Garfinkel, Christine E Seidman
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful cellular platform for illuminating mechanisms of human cardiovascular disease and for pharmacological screening. Recent advances in CRISPR/Cas9-mediated genome editing technology underlie this profound utility. We have generated hiPSC-CMs harboring fluorescently-tagged sarcomeric proteins, which provide a tool to non-invasively study human sarcomere function and dysfunction. In this unit, we illustrate methods for conducting high-efficiency, small molecule-mediated differentiation of hiPSCs into cardiomyocytes, and for performing non-invasive contractile analysis through direct sarcomere tracking of GFP-sarcomere reporter hiPSC-CMs...
January 24, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29364521/biosafety-in-handling-gene-transfer-vectors
#13
Scott Swindle
This unit is devoted to safety issues that must be considered when generating and working with the most common vectors under development for human gene therapy today. © 2018 by John Wiley & Sons, Inc.
January 24, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29364520/balanced-chromosomal-rearrangement-detection-by-low-pass-whole-genome-sequencing
#14
Zirui Dong, Lingfei Ye, Zhenjun Yang, Haixiao Chen, Jianying Yuan, Huilin Wang, Xiaosen Guo, Yun Li, Jun Wang, Fang Chen, Sau Wai Cheung, Cynthia C Morton, Hui Jiang, Kwong Wai Choy
Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole-genome sequencing (WGS) for detection of BCA-associated breakpoints, but the requirement for a priori knowledge of the rearranged regions from G-banded chromosome analysis limits its application. The protocols described here are based on low-pass WGS for detecting BCA events independent from chromosome analysis, and has been validated using genomic data from the 1000 Genomes Project...
January 24, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29364519/crispr-cas9-mediated-fluorescent-tagging-of-endogenous-proteins-in-human-pluripotent-stem-cells
#15
Arun Sharma, Christopher N Toepfer, Tarsha Ward, Lauren Wasson, Radhika Agarwal, David A Conner, Johnny H Hu, Christine E Seidman
Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length...
January 24, 2018: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29044473/induced-pluripotent-stem-cells-from-ovarian-tissue
#16
Sophia Salas, Nicholas Ng, Behzad Gerami-Naini, Raymond M Anchan
Yamanaka and colleagues revolutionized stem cell biology and regenerative medicine by observing that somatic cells can be reprogrammed into pluripotent stem cells. Evidence indicates that induced pluripotent stem (iPS) cells retain epigenetic memories that bias their spontaneous differentiation into the originating somatic cell type, therefore epigenetic memory may be exploited to improve tissue specific regeneration. We recently showed that iPS cells reprogrammed from ovarian granulosa cells using mouse and human tissue overwhelmingly differentiate homotypically into ovarian steroidogenic and primordial germ cells...
October 18, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29044472/population-stratification-in-genetic-association-studies
#17
REVIEW
Jacklyn N Hellwege, Jacob M Keaton, Ayush Giri, Xiaoyi Gao, Digna R Velez Edwards, Todd L Edwards
Population stratification (PS) is a primary consideration in studies of genetic determinants of human traits. Failure to control for PS may lead to confounding, causing a study to fail for lack of significant results, or resources to be wasted following false-positive signals. Here, historical and current approaches for addressing PS when performing genetic association studies in human populations are reviewed. Methods for detecting the presence of PS, including global and local ancestry methods, are described...
October 18, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29044471/analysis-and-annotation-of-whole-genome-or-whole-exome-sequencing-derived-variants-for-clinical-diagnosis
#18
Elizabeth A Worthey
Over the last 10 years, next-generation sequencing (NGS) has transformed genomic research through substantial advances in technology and reduction in the cost of sequencing, and also in the systems required for analysis of these large volumes of data. This technology is now being used as a standard molecular diagnostic test in some clinical settings. The advances in sequencing have come so rapidly that the major bottleneck in identification of causal variants is no longer the sequencing or analysis (given access to appropriate tools), but rather clinical interpretation...
October 18, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29044470/analysis-of-gene-gene-interactions
#19
REVIEW
Brian S Cole, Molly A Hall, Ryan J Urbanowicz, Diane Gilbert-Diamond, Jason H Moore
The goal of this unit is to introduce epistasis, or gene-gene interactions, as a significant contributor to the genetic architecture of complex traits, including disease susceptibility. This unit begins with an historical overview of the concept of epistasis and the challenges inherent in the identification of potential gene-gene interactions. Then, it reviews statistical and machine learning methods for discovering epistasis in the context of genetic studies of quantitative and categorical traits. This unit concludes with a discussion of meta-analysis, replication, and other topics of active research...
October 18, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/29044469/co-differentiation-of-human-pluripotent-stem-cells-derived-cardiomyocytes-and-endothelial-cells-from-cardiac-mesoderm-provides-a-three-dimensional-model-of-cardiac-microtissue
#20
Elisa Giacomelli, Milena Bellin, Valeria V Orlova, Christine L Mummery
The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPS cells) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular progenitors in human embryos. The generation of cardiac endothelial cells in particular allows the creation of complex models of cardiovascular disorders in which either cardiomyocytes or endothelial cells are affected...
October 18, 2017: Current Protocols in Human Genetics
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