Current Protocols in Human Genetics最新文献

Resolving Breakpoints of Chromosomal Rearrangements at the Nucleotide Level Using Sanger Sequencing 利用Sanger测序在核苷酸水平上解决染色体重排的断裂点。

Current Protocols in Human Genetics Pub Date : 2020-12-23 DOI: 10.1002/cphg.107

Katarena Nalbandian, Raul E. Piña-Aguilar, Cynthia C. Morton

{"title":"Resolving Breakpoints of Chromosomal Rearrangements at the Nucleotide Level Using Sanger Sequencing","authors":"Katarena Nalbandian, Raul E. Piña-Aguilar, Cynthia C. Morton","doi":"10.1002/cphg.107","DOIUrl":"10.1002/cphg.107","url":null,"abstract":"Novel cytogenetic tools are increasingly based on genome sequencing for detecting chromosomal abnormalities. Different sequence-based techniques optimized for diagnosis of structural variants can be useful for narrowing down the localization of breakpoints of chromosomal abnormalities, but do not offer nucleotide resolution of breakpoints for proper interpretation of gene disruption. This protocol presents the characterization of structural variants at nucleotide resolution using Sanger sequencing after low-pass large-insert genome sequencing or other long-molecule methods. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Primer design for junction amplification at translocations and inversionsBasic Protocol 2: Amplification of derivative chromosomes using a long-range polymeraseAlternate Protocol: Amplification of derivative chromosomes using a hot-start polymeraseBasic Protocol 3: Preparation of DNA for Sanger sequencingBasic Protocol 4: Interpretation and reporting of breakpoints based on Sanger sequencing","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39092313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Informed Consent for Genetic and Genomic Research 遗传和基因组研究的知情同意

Current Protocols in Human Genetics Pub Date : 2020-11-17 DOI: 10.1002/cphg.104

Jeffrey R. Botkin

引用次数: 0

A Guide to Using ClinTAD for Interpretation of DNA Copy Number Variants in the Context of Topologically Associated Domains 使用ClinTAD在拓扑相关域的背景下解释DNA拷贝数变异的指南

Current Protocols in Human Genetics Pub Date : 2020-11-10 DOI: 10.1002/cphg.106

Jacob D. Spector, Arun P. Wiita

{"title":"A Guide to Using ClinTAD for Interpretation of DNA Copy Number Variants in the Context of Topologically Associated Domains","authors":"Jacob D. Spector, Arun P. Wiita","doi":"10.1002/cphg.106","DOIUrl":"10.1002/cphg.106","url":null,"abstract":"DNA copy number variants (CNVs) are routinely evaluated as part of clinical diagnosis in both the prenatal and postnatal genetic settings. Current guidelines for interpreting the potential clinical significance of these CNVs, typically identified by chromosomal microarray, focus entirely on genes localized within the CNV region. However, recent work has suggested that some CNVs can actually produce clinical impacts by influencing transcription of genes outside the CNV region. These alterations of transcription appear to occur by disrupting the composition of DNA topologically associated domains (TADs), which strongly influence contacts between gene promoters and their associated enhancers. Here we present a set of detailed protocols for the use of the free software tool ClinTAD (https://www.clintad.com). This decision‐support software allows for prediction as to whether a given CNV may potentially disrupt a TAD boundary, and offers phenotype matching to genes near, but not within the CNV region, whose expression could be influenced by altered TAD architecture and that have phenotypic impacts related to that reported in a given patient. Our protocols here provide specific examples of how to implement these tools. In addition, the software has the capability to impact genomic research by evaluating multiple cases in parallel. We propose that this decision‐support tool can benefit and improve genetic diagnosis. © 2020 Wiley Periodicals LLC.","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38585475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The AD Knowledge Portal: A Repository for Multi-Omic Data on Alzheimer's Disease and Aging AD知识门户:关于阿尔茨海默病和衰老的多组学数据储存库

Current Protocols in Human Genetics Pub Date : 2020-10-21 DOI: 10.1002/cphg.105

Anna K. Greenwood, Kelsey S. Montgomery, Nicole Kauer, Kara H. Woo, Zoe J. Leanza, William L. Poehlman, Jake Gockley, Solveig K. Sieberts, Ljubomir Bradic, Benjamin A. Logsdon, Mette A. Peters, Larsson Omberg, Lara M. Mangravite

{"title":"The AD Knowledge Portal: A Repository for Multi-Omic Data on Alzheimer's Disease and Aging","authors":"Anna K. Greenwood, Kelsey S. Montgomery, Nicole Kauer, Kara H. Woo, Zoe J. Leanza, William L. Poehlman, Jake Gockley, Solveig K. Sieberts, Ljubomir Bradic, Benjamin A. Logsdon, Mette A. Peters, Larsson Omberg, Lara M. Mangravite","doi":"10.1002/cphg.105","DOIUrl":"10.1002/cphg.105","url":null,"abstract":"The AD Knowledge Portal (adknowledgeportal.org) is a public data repository that shares data and other resources generated by multiple collaborative research programs focused on aging, dementia, and Alzheimer's disease (AD). In this article, we highlight how to use the Portal to discover and download genomic variant and transcriptomic data from the same individuals. First, we show how to use the web interface to browse and search for data of interest using relevant file annotations. We demonstrate how to learn more about the context surrounding the data, including diagnostic criteria and methodological details about sample preparation and data analysis. We present two primary ways to download data—using a web interface, and using a programmatic method that provides access using the command line. Finally, we show how to merge separate sources of metadata into a comprehensive file that contains factors and covariates necessary in downstream analyses. © 2020 The Authors.Basic Protocol 1: Find and download files associated with a selected studyBasic Protocol 2: Download files in bulk using the command line clientBasic Protocol 3: Working with file annotations and metadata","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38616356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 26

A Practical Guide for Structural Variation Detection in the Human Genome 人类基因组结构变异检测实用指南。

Current Protocols in Human Genetics Pub Date : 2020-08-19 DOI: 10.1002/cphg.103

Lixing Yang

{"title":"A Practical Guide for Structural Variation Detection in the Human Genome","authors":"Lixing Yang","doi":"10.1002/cphg.103","DOIUrl":"10.1002/cphg.103","url":null,"abstract":"Profiling genetic variants—including single nucleotide variants, small insertions and deletions, copy number variations, and structural variations (SVs)—from both healthy individuals and individuals with disease is a key component of genetic and biomedical research. SVs are large-scale changes in the genome and involve breakage and rejoining of DNA fragments. They may affect thousands to millions of nucleotides and can lead to loss, gain, and reshuffling of genes and regulatory elements. SVs are known to impact gene expression and potentially result in altered phenotypes and diseases. Therefore, identifying SVs from the human genomes is particularly important. In this review, I describe advantages and disadvantages of the available high-throughput assays for the discovery of SVs, which are the most challenging genetic alterations to detect. A practical guide is offered to suggest the most suitable strategies for discovering different types of SVs including common germline, rare, somatic, and complex variants. I also discuss factors to be considered, such as cost and performance, for different strategies when designing experiments. Last, I present several approaches to identify potential SV artifacts caused by samples, experimental procedures, and computational analysis. © 2020 Wiley Periodicals LLC.","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"107 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38279777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Identification and Genotyping of Transposable Element Insertions From Genome Sequencing Data 基因组测序数据中转座元件插入的鉴定和基因分型

Current Protocols in Human Genetics Pub Date : 2020-07-14 DOI: 10.1002/cphg.102

Chong Chu, Boxun Zhao, Peter J. Park, Eunjung Alice Lee

引用次数: 8

Analytical Approaches for ATAC-seq Data Analysis ATAC-seq数据分析方法

Current Protocols in Human Genetics Pub Date : 2020-06-15 DOI: 10.1002/cphg.101

Jason P. Smith, Nathan C. Sheffield

引用次数: 11

Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro 将扩展的三核苷酸重复序列引入人类基因组用于体外亨廷顿氏病建模

Current Protocols in Human Genetics Pub Date : 2020-05-29 DOI: 10.1002/cphg.100

Tuyana Malankhanova, Michael Sorokin, Sergey Medvedev, Suren Zakian, Anastasia Malakhova

{"title":"Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro","authors":"Tuyana Malankhanova, Michael Sorokin, Sergey Medvedev, Suren Zakian, Anastasia Malakhova","doi":"10.1002/cphg.100","DOIUrl":"10.1002/cphg.100","url":null,"abstract":"In neurodegeneration studies, researchers are faced with problems such as limited material availability and late disease manifestation. Cell models provide the opportunity to investigate molecular mechanisms of pathogenesis. Moreover, genome editing technologies enable generation of isogenic cell models of hereditary diseases. Our protocol outlines an approach for introducing an expanded CAG repeat tract into the first exon of the HTT gene, the Huntington's disease causing mutation. The protocol allows modeling the disease at various severity levels by introducing different numbers of CAG repeats. Furthermore, the protocol can be applicable for modeling other diseases caused by trinucleotide repeat expansion. It is important to note there are many difficulties with cloning repeated sequences and amplification of GC-rich regions. Here, we also propose troubleshooting options, which overcome these problems. The protocol is based on CRISPR/Cas9-mediated homologous recombination with a uniquely designed donor plasmid harboring an expanded CAG tract flanked with long homology arms. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Design and assembling donor and CRISPR/Cas9-expressing plasmidsBasic Protocol 2: Transfection of cells with plasmids and sorting GFP-positive cellsBasic Protocol 3: PCR screening single-cell clones and validation of the mutant HTT expression","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37985978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Clinical Interpretation of Sequence Variants 序列变异的临床解释。

Current Protocols in Human Genetics Pub Date : 2020-03-16 DOI: 10.1002/cphg.98

Junyu Zhang, Yanyi Yao, Haixian He, Jun Shen

{"title":"Clinical Interpretation of Sequence Variants","authors":"Junyu Zhang, Yanyi Yao, Haixian He, Jun Shen","doi":"10.1002/cphg.98","DOIUrl":"10.1002/cphg.98","url":null,"abstract":"Clinical interpretation of DNA sequence variants is a critical step in reporting clinical genetic testing results. Application of next-generation sequencing technology in molecular genetic testing has facilitated diagnoses of genetic disorders in clinical practice. However, the large number of DNA sequence variants detected in clinical specimens, many of which have never been seen before, make clinical interpretation challenging. Recommendations by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) have been widely adopted by clinical laboratories around the world to guide clinical interpretation of sequence variants. The ClinGen Sequence Variant Interpretation Working Group and various disease-specific variant curation expert panels have also developed specifications for the ACMG/AMP recommendations. Despite these efforts to standardize variant interpretation in clinical practice, different laboratories may subjectively use professional judgment to determine which criteria are applicable when classifying a variant. In addition, clinicians and researchers who are not familiar with the variant interpretation process may have difficulty understanding clinical genetic reports and communicating the clinical significance of genetic testing results. Here we provide a step-by-step protocol for clinical interpretation of sequence variants, including practical examples. By following this protocol, clinical laboratory geneticists can interpret the clinical significance of sequence variants according to the ACMG/AMP recommendations and ClinGen framework. Furthermore, this article will help clinicians and researchers to understand variant classification in clinical genetic testing reports and evaluate the quality of the reports. © 2020 by John Wiley & Sons, Inc.Basic Protocol: Interpreting the clinical significance of sequence variantsSupport Protocol: Reevaluating the clinical significance of sequence variants","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.98","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37742236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 40

Quantitative Assessment of Parental Somatic Mosaicism for Copy-Number Variant (CNV) Deletions 拷贝数变异(CNV)缺失对亲本体细胞嵌合现象的定量评价

Current Protocols in Human Genetics Pub Date : 2020-03-16 DOI: 10.1002/cphg.99

Qian Liu, Christopher M. Grochowski, Weimin Bi, James R. Lupski, Paweł Stankiewicz

{"title":"Quantitative Assessment of Parental Somatic Mosaicism for Copy-Number Variant (CNV) Deletions","authors":"Qian Liu, Christopher M. Grochowski, Weimin Bi, James R. Lupski, Paweł Stankiewicz","doi":"10.1002/cphg.99","DOIUrl":"10.1002/cphg.99","url":null,"abstract":"As genome sequencing methodologies have become more sensitive in detecting low-frequency rare-variant events, the link between post-zygotic mutagenesis and somatic mosaicism in the etiology of several human genetic conditions other than cancers has become more clear. Given that current clinical-genomics diagnostic methods have limited detection sensitivity for mosaic events, a copy-number variant (CNV) deletion inherited from a parent with low-level (<10%) mosaicism can be erroneously interpreted in the proband to represent a de novo germline event. Here, we describe three sensitive, precise, and cost-efficient methods that can quantitatively assess the potential degree of parental somatic mosaicism levels for CNV deletions: droplet digital PCR (ddPCR), PCR amplicon–based next-generation sequencing (NGS), and quantitative PCR. ddPCR using the EvaGreen fluorescent dye protocol can specifically quantify the deleted or non-deleted alleles by analyzing the number of droplets positive for a fluorescent signal for each event. PCR amplicon–based NGS assesses the allele frequencies of a heterozygous single-nucleotide polymorphism within a deletion region. The difference in number of reads between the two genotypes indicates the level of somatic mosaicism for the CNV deletion. Quantitative PCR can be applied where the relative quantity of the deletion junction–specific product represents the level of mosaicism. Clinical implementation of these quantitative variant-detection methods enables potentially more accurate assessment of disease recurrence risk in family-based genetic counseling, allowing couples to engage in more informed family planning. © 2020 by John Wiley & Sons, Inc.Basic Protocol: Droplet digital PCR (ddPCR)Alternate Protocol 1: PCR amplicon–based next-generation sequencingAlternate Protocol 2: Quantitative real-time PCR (qPCR)","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"106 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.99","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37742235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9