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Current Protocols in Human Genetics最新文献

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Balanced Chromosomal Rearrangement Detection by Low-Pass Whole-Genome Sequencing 平衡染色体重排检测低通全基因组测序
Current Protocols in Human Genetics Pub Date : 2018-01-24 DOI: 10.1002/cphg.51
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
{"title":"Balanced Chromosomal Rearrangement Detection by Low-Pass Whole-Genome Sequencing","authors":"Zirui Dong,&nbsp;Lingfei Ye,&nbsp;Zhenjun Yang,&nbsp;Haixiao Chen,&nbsp;Jianying Yuan,&nbsp;Huilin Wang,&nbsp;Xiaosen Guo,&nbsp;Yun Li,&nbsp;Jun Wang,&nbsp;Fang Chen,&nbsp;Sau Wai Cheung,&nbsp;Cynthia C. Morton,&nbsp;Hui Jiang,&nbsp;Kwong Wai Choy","doi":"10.1002/cphg.51","DOIUrl":"10.1002/cphg.51","url":null,"abstract":"<p>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 <i>a priori</i> 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. This approach adopts non-size-selected mate-pair library (3∼8 kb) with 2∼3 μg DNA as input, and requires only 30 million read-pairs (50 bp, equivalent to 1-fold base-coverage) for each sample. The complete procedure takes 13 days and the total cost is estimated to be less than $600 (USD) per sample. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.51","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35763627","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: 13
Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes gfp -肌节报告细胞hipsc -心肌细胞的分化和收缩分析
Current Protocols in Human Genetics Pub Date : 2018-01-24 DOI: 10.1002/cphg.53
Arun Sharma, Christopher N. Toepfer, Manuel Schmid, Amanda C. Garfinkel, Christine E. Seidman
{"title":"Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes","authors":"Arun Sharma,&nbsp;Christopher N. Toepfer,&nbsp;Manuel Schmid,&nbsp;Amanda C. Garfinkel,&nbsp;Christine E. Seidman","doi":"10.1002/cphg.53","DOIUrl":"10.1002/cphg.53","url":null,"abstract":"<p>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. We believe that this type of analysis can overcome sensitivity problems found in other forms of contractile assays involving hiPSC-CMs by directly measuring contractility at the fundamental contractile unit of the hiPSC-CM, the sarcomere. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.53","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35762574","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: 27
Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC) 遗传性非息肉病性结直肠癌的基因检测
Current Protocols in Human Genetics Pub Date : 2017-07-11 DOI: 10.1002/cphg.40
Babi Ramesh Reddy Nallamilli, Madhuri Hegde
{"title":"Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)","authors":"Babi Ramesh Reddy Nallamilli,&nbsp;Madhuri Hegde","doi":"10.1002/cphg.40","DOIUrl":"10.1002/cphg.40","url":null,"abstract":"<p>Hereditary nonpolyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominant cancer syndrome that confers an elevated risk of early-onset colorectal cancer (CRC) and increased lifetime risk for other cancers of the endometrium, stomach, small intestine, hepatobiliary system, kidney, ureter, and ovary. Lynch syndrome accounts for up to 3% of all CRC, making it the most common hereditary colorectal cancer syndrome. Germline mutations in methyl-directed mismatch repair (MMR) genes give rise to microsatellite instability (MSI) in tumor DNA. Lynch syndrome is most frequently caused by pathogrenic variants in the mismatch repair genes <i>MLH1</i>, <i>MSH2</i>, <i>MSH6</i>, and <i>PMS2</i>. Germline mutations in <i>MLH1</i> and <i>MSH2</i> account for approximately 90% of detected mutations in families with Lynch syndrome. Pathogenic vatiants in <i>MSH6</i> have been reported in approximately 7-10% of families with Lynch syndrome. Pathogenic variants in <i>PMS2</i> account for fewer than 5% of mutations in families with Lynch syndrome. This unit presents a comprehensive molecular genetic testing strategy for Lynch syndrome including MSI analysis, next generation sequencing (NGS)-based targeted sequence analysis, PCR-based Sanger sequencing and microarray-based comparative genomic hybridization (array-CGH). © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35158822","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: 11
Copy-Number Variants Detection by Low-Pass Whole-Genome Sequencing 低通全基因组测序的拷贝数变异检测
Current Protocols in Human Genetics Pub Date : 2017-07-11 DOI: 10.1002/cphg.43
Zirui Dong, Weiwei Xie, Haixiao Chen, Jinjin Xu, Huilin Wang, Yun Li, Jun Wang, Fang Chen, Kwong Wai Choy, Hui Jiang
{"title":"Copy-Number Variants Detection by Low-Pass Whole-Genome Sequencing","authors":"Zirui Dong,&nbsp;Weiwei Xie,&nbsp;Haixiao Chen,&nbsp;Jinjin Xu,&nbsp;Huilin Wang,&nbsp;Yun Li,&nbsp;Jun Wang,&nbsp;Fang Chen,&nbsp;Kwong Wai Choy,&nbsp;Hui Jiang","doi":"10.1002/cphg.43","DOIUrl":"10.1002/cphg.43","url":null,"abstract":"<p>Emerging studies have demonstrated that whole-genome sequencing (WGS) is an efficient tool for copy-number variants (CNV) detection, particularly in probe-poor regions, as compared to chromosomal microarray analysis (CMA). However, the cost of testing is beyond economical for routine usage and the lengthy turn-around time is not ideal for clinical implementation. In addition, the demand for computational resources also reduces the probability of clinical integration into each laboratory. Herein, a protocol providing CNV detection from low-pass, whole-genome sequencing (0.25×) in a clinical laboratory setting is described. The cost is reduced to less than $200 USD per sample and the turn-around time is within an acceptable clinically workable time-frame (7 days). © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.43","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35158818","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: 23
Overview of Admixture Mapping 外加剂映射概述
Current Protocols in Human Genetics Pub Date : 2017-07-11 DOI: 10.1002/cphg.44
Daniel Shriner
{"title":"Overview of Admixture Mapping","authors":"Daniel Shriner","doi":"10.1002/cphg.44","DOIUrl":"10.1002/cphg.44","url":null,"abstract":"<p>Admixture mapping is a powerful method of gene mapping for diseases or traits that show differential risk by ancestry. Admixture mapping has been applied most often to Americans who trace ancestry to various combinations of Native Americans, Europeans, and West Africans. Recent developments in admixture mapping include improvements in methods and the reference data needed to make inferences about ancestry, as well as extensions of the mapping approach in the framework of linear mixed models. In this unit, the key concepts of admixture mapping are outlined. Several approaches for inferring local ancestry are described, and strategies for performing admixture mapping depending on the study design are provided. Finally, comparisons and contrasts between linkage analysis, association analysis, and admixture mapping are provided, with an emphasis on integrating admixture mapping and association testing. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.44","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35158823","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: 65
1D Genome Sequencing on the Oxford Nanopore MinION 牛津纳米孔MinION的1D基因组测序
Current Protocols in Human Genetics Pub Date : 2017-07-11 DOI: 10.1002/cphg.39
Sara Goodwin, Robert Wappel, W. Richard McCombie
{"title":"1D Genome Sequencing on the Oxford Nanopore MinION","authors":"Sara Goodwin,&nbsp;Robert Wappel,&nbsp;W. Richard McCombie","doi":"10.1002/cphg.39","DOIUrl":"10.1002/cphg.39","url":null,"abstract":"<p>Today's short-read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high-throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become indispensable tools for many researchers. With greater insight has come the revelation that many genomes are much more complicated than originally thought and include many rearrangements and copy-number variations. Unfortunately, short-read sequencing technologies are not well suited for identifying many of these types of events. Long-read sequencing technologies can read contiguous fragments of DNA in excess of 10 kb and are much better suited for detecting large structural events. The newest long-read sequencing instrument is the MinION device from Oxford Nanopore. The rapid sequencing speed and low upfront instrument cost are features drawing interest in this device from the genomics community. This unit provides a representative protocol for carrying out human genome sequencing on the Oxford Nanopore MinION. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.39","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35158819","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: 7
Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas 非霍奇金淋巴瘤遗传标记的分子分析
Current Protocols in Human Genetics Pub Date : 2017-04-06 DOI: 10.1002/cphg.37
Lynette M. Sholl, Janina Longtine, Frank C. Kuo
{"title":"Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas","authors":"Lynette M. Sholl,&nbsp;Janina Longtine,&nbsp;Frank C. Kuo","doi":"10.1002/cphg.37","DOIUrl":"10.1002/cphg.37","url":null,"abstract":"<p>Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen-receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.37","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34892129","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: 3
Methods for Quantitative Creatinine Determination 定量测定肌酐的方法
Current Protocols in Human Genetics Pub Date : 2017-04-06 DOI: 10.1002/cphg.38
John F. Moore, J. Daniel Sharer
{"title":"Methods for Quantitative Creatinine Determination","authors":"John F. Moore,&nbsp;J. Daniel Sharer","doi":"10.1002/cphg.38","DOIUrl":"10.1002/cphg.38","url":null,"abstract":"<p>Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine-picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well as a modified, automated version. Also described is a high performance liquid chromatography (HPLC) method that separates creatinine from contaminants prior to direct quantification by UV absorption. Lastly, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that uses stable isotope dilution to reliably quantify creatinine in any sample. This last approach has been recommended by experts in the field as a means to standardize all quantitative creatinine methods against an accepted reference. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34892128","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: 35
Overview of Genetic Diagnosis in Cancer 癌症基因诊断综述
Current Protocols in Human Genetics Pub Date : 2017-04-06 DOI: 10.1002/cphg.36
Bruce R. Korf, Fady M. Mikhail
{"title":"Overview of Genetic Diagnosis in Cancer","authors":"Bruce R. Korf,&nbsp;Fady M. Mikhail","doi":"10.1002/cphg.36","DOIUrl":"10.1002/cphg.36","url":null,"abstract":"<p>Both cytogenetic and molecular genetic studies can contribute to the management of patients with cancer. In some cases, genetic markers are specific to particular tumor types and are useful in diagnosis. This can be helpful in distinguishing histologically similar tumors that may respond differently to treatment and can sometimes be of prognostic value. Genetic markers can also be tools for following the response of a tumor to therapy, providing a sensitive means to detect relapse. This introductory unit considers some of the types of genetic changes that occur in association with malignancies, as well as major approaches used in their detection. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.36","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34892130","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: 2
Computational Approach to Measuring Myocyte Disarray in Animal Models of Heart Disease 测量心脏疾病动物模型中心肌细胞紊乱的计算方法
Current Protocols in Human Genetics Pub Date : 2017-04-06 DOI: 10.1002/cphg.35
William Wan, Leslie Leinwand
{"title":"Computational Approach to Measuring Myocyte Disarray in Animal Models of Heart Disease","authors":"William Wan,&nbsp;Leslie Leinwand","doi":"10.1002/cphg.35","DOIUrl":"10.1002/cphg.35","url":null,"abstract":"<p>In cardiovascular disease research, studies often include measuring cardiac function and performing histological examination of heart tissue. After measuring contractility, hearts from animals such as mice and rats are often frozen or fixed, sliced, and stained to quantify the morphology of various structures such as extracellular matrix proteins, cell nuclei, and F-actin. Traditional scoring methods have largely consisted of assessing sections of images for the presence or absence of myocyte disarray. These approaches require unbiased manual assessment, which can require extra personnel, and are not scalable to the quantity of data that can be generated by modern automated experimental techniques. Here, we describe an automated image analysis approach for unbiased numerical measurement of myocyte disarray. We provide step-by-step instructions for image preparation as well as a basic Matlab script for measurements. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40007,"journal":{"name":"Current Protocols in Human Genetics","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cphg.35","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34892131","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
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