Current Protocols in Human Genetics最新文献

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 inversions

Basic Protocol 2: Amplification of derivative chromosomes using a long-range polymerase

Alternate Protocol: Amplification of derivative chromosomes using a hot-start polymerase

Basic Protocol 3: Preparation of DNA for Sanger sequencing

Basic Protocol 4: Interpretation and reporting of breakpoints based on Sanger sequencing

Genetic research often utilizes or generates information that is potentially sensitive to individuals, families, or communities. For these reasons, genetic research may warrant additional scrutiny from investigators and governmental regulators, compared to other types of biomedical research. The informed consent process should address the range of social and psychological issues that may arise in genetic research. This article addresses a number of these issues, including recruitment of participants, disclosure of results, psychological impact of results, insurance and employment discrimination, community engagement, consent for tissue banking, and intellectual property issues. Points of consideration are offered to assist in the development of protocols and consent processes in light of contemporary debates on a number of these issues. © 2020 Wiley Periodicals LLC.

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 study

Basic Protocol 2: Download files in bulk using the command line client

Basic Protocol 3: Working with file annotations and metadata

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.

Transposable element (TE) mobilization is a significant source of genomic variation and has been associated with various human diseases. The exponential growth of population-scale whole-genome sequencing and rapid innovations in long-read sequencing technologies provide unprecedented opportunities to study TE insertions and their functional impact in human health and disease. Identifying TE insertions, however, is challenging due to the repetitive nature of the TE sequences. Here, we review computational approaches to detecting and genotyping TE insertions using short- and long-read sequencing and discuss the strengths and weaknesses of different approaches. © 2020 Wiley Periodicals LLC.

ATAC-seq, the assay for transposase-accessible chromatin using sequencing, is a quick and efficient approach to investigating the chromatin accessibility landscape. Investigating chromatin accessibility has broad utility for answering many biological questions, such as mapping nucleosomes, identifying transcription factor binding sites, and measuring differential activity of DNA regulatory elements. Because the ATAC-seq protocol is both simple and relatively inexpensive, there has been a rapid increase in the availability of chromatin accessibility data. Furthermore, advances in ATAC-seq protocols are rapidly extending its breadth to additional experimental conditions, cell types, and species. Accompanying the increase in data, there has also been an explosion of new tools and analytical approaches for analyzing it. Here, we explain the fundamentals of ATAC-seq data processing, summarize common analysis approaches, and review computational tools to provide recommendations for different research questions. This primer provides a starting point and a reference for analysis of ATAC-seq data. © 2020 Wiley Periodicals LLC.

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 plasmids

Basic Protocol 2: Transfection of cells with plasmids and sorting GFP-positive cells

Basic Protocol 3: PCR screening single-cell clones and validation of the mutant HTT expression

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 variants

Support Protocol: Reevaluating the clinical significance of sequence variants

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 sequencing

Alternate Protocol 2: Quantitative real-time PCR (qPCR)