scispace - formally typeset
Search or ask a question

Showing papers in "Current protocols in human genetics in 2013"


Journal ArticleDOI
TL;DR: This unit describes how to use BWA and the Genome Analysis Toolkit to map genome sequencing data to a reference and produce high‐quality variant calls that can be used in downstream analyses.
Abstract: This unit describes how to use BWA and the Genome Analysis Toolkit (GATK) to map genome sequencing data to a reference and produce high-quality variant calls that can be used in downstream analyses. The complete workflow includes the core NGS data processing steps that are necessary to make the raw data suitable for analysis by the GATK, as well as the key methods involved in variant discovery using the GATK.

5,150 citations


Journal ArticleDOI
TL;DR: PolyPhen‐2 (Polymorphism Phenotyping v2), available as software and via a Web server, predicts the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations.
Abstract: PolyPhen-2 (Polymorphism Phenotyping v2), available as software and via a Web server, predicts the possible impact of amino acid substitutions on the stability and function of human proteins using structural and comparative evolutionary considerations. It performs functional annotation of single-nucleotide polymorphisms (SNPs), maps coding SNPs to gene transcripts, extracts protein sequence annotations and structural attributes, and builds conservation profiles. It then estimates the probability of the missense mutation being damaging based on a combination of all these properties. PolyPhen-2 features include a high-quality multiple protein sequence alignment pipeline and a prediction method employing machine-learning classification. The software also integrates the UCSC Genome Browser's human genome annotations and MultiZ multiple alignments of vertebrate genomes with the human genome. PolyPhen-2 is capable of analyzing large volumes of data produced by next-generation sequencing projects, thanks to built-in support for high-performance computing environments like Grid Engine and Platform LSF.

2,681 citations


Journal ArticleDOI
TL;DR: This unit provides an overview of the inference of homology from significant similarity, and introduces other units in this chapter that provide more details on effective strategies for identifying homologs.
Abstract: Sequence similarity searching, typically with BLAST, is the most widely used and most reliable strategy for characterizing newly determined sequences. Sequence similarity searches can identify "homologous" proteins or genes by detecting excess similarity- statistically significant similarity that reflects common ancestry. This unit provides an overview of the inference of homology from significant similarity, and introduces other units in this chapter that provide more details on effective strategies for identifying homologs.

490 citations


Journal ArticleDOI
TL;DR: The Mitomap protocol shows how users may look up human mitochondrial gene loci, search for public mitochondrial sequences, and browse or search for reported general population nucleotide variants as well as those reported in clinical disease.
Abstract: The Mitomap database of human mitochondrial DNA (mtDNA) information has been an important compilation of mtDNA variation for researchers, clinicians and genetic counselors for the past twenty-five years. The Mitomap protocol shows how users may look up human mitochondrial gene loci, search for public mitochondrial sequences, and browse or search for reported general population nucleotide variants as well as those reported in clinical disease. Within Mitomap is the powerful sequence analysis tool for human mitochondrial DNA, Mitomaster. The Mitomaster protocol gives step-by-step instructions showing how to submit sequences to identify nucleotide variants relative to the rCRS, to determine the haplogroup, and to view species conservation. User-supplied sequences, GenBank identifiers and single nucleotide variants may be analyzed.

417 citations


Journal ArticleDOI
TL;DR: VarScan 2 as discussed by the authors employs heuristic and statistic thresholds based on user-defined criteria to call variants using SAMtools mpileup data as input, and describes protocols for using VarScan 2 to identify germline variants in individual samples, call somatic mutations, copy number alterations, and LOH events in tumor-normal pairs.
Abstract: The identification of small sequence variants remains a challenging but critical step in the analysis of next-generation sequencing data Our variant calling tool, VarScan 2, employs heuristic and statistic thresholds based on user-defined criteria to call variants using SAMtools mpileup data as input Here, we provide guidelines for generating that input, and describe protocols for using VarScan 2 to (1) identify germline variants in individual samples; (2) call somatic mutations, copy number alterations, and LOH events in tumor-normal pairs; and (3) identify germline variants, de novo mutations, and Mendelian inheritance errors in family trios Further, we describe a strategy for variant filtering that removes likely false positives associated with common sequencing- and alignment-related artifacts

187 citations


Journal ArticleDOI
TL;DR: Theoretical foundations that drive practical choices of protein and DNA similarity scoring matrices and gap penalties are discussed, including the role of match and mismatch parameters in DNA searches.
Abstract: Protein sequence similarity searching programs like BLASTP, SSEARCH (UNIT 3.10), and FASTA use scoring matrices that are designed to identify distant evolutionary relationships (BLOSUM62 for BLAST, BLOSUM50 for SEARCH and FASTA). Different similarity scoring matrices are most effective at different evolutionary distances. "Deep" scoring matrices like BLOSUM62 and BLOSUM50 target alignments with 20 - 30% identity, while "shallow" scoring matrices (e.g. VTML10 - VTML80), target alignments that share 90 - 50% identity, reflecting much less evolutionary change. While "deep" matrices provide very sensitive similarity searches, they also require longer sequence alignments and can sometimes produce alignment overextension into non-homologous regions. Shallower scoring matrices are more effective when searching for short protein domains, or when the goal is to limit the scope of the search to sequences that are likely to be orthologous between recently diverged organisms. Likewise, in DNA searches, the match and mismatch parameters set evolutionary look-back times and domain boundaries. In this unit, we will discuss the theoretical foundations that drive practical choices of protein and DNA similarity scoring matrices and gap penalties. Deep scoring matrices (BLOSUM62 and BLOSUM50) should be used for sensitive searches with full-length protein sequences, but short domains or restricted evolutionary look-back require shallower scoring matrices.

118 citations


Reference EntryDOI
TL;DR: A set of improvements that have been made to the standard Illumina protocols to make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data are described.
Abstract: In this unit, we describe a set of improvements that have been made to the standard Illumina protocols to make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data.

116 citations


Journal ArticleDOI
TL;DR: The author describes several approaches for inferring local ancestry and provides strategies for performing admixtures mapping depending on the study design, and compares and contrasts linkage analysis, association analysis, and admixture mapping.
Abstract: 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 & Sons, Inc.

76 citations


Journal ArticleDOI
TL;DR: This unit shows how to use the SOAPaligner package to align short reads to reference, and includes a protocol for SNP calling from SOAP2 alignment with SOAPsnp.
Abstract: This unit shows how to use the SOAPaligner package to align short reads to reference. The use of the two most common versions of SOAPaligner, SOAP2 and SOAP3-dp, will be described in detail. The unit also includes a protocol for SNP calling from SOAP2 alignment with SOAPsnp.

59 citations


Journal ArticleDOI
TL;DR: The software supports batch processing of multiple shotgun acquisitions by high‐resolution mass mapping, precursor and neutral‐loss scanning, and data‐dependent MS/MS lending itself to a variety of lipidomics applications in cell biology and molecular medicine.
Abstract: LipidXplorer is an open-source software kit that supports the identification and quantification of molecular species of any lipid class detected by shotgun experiments performed on any mass spectrometry platform. LipidXplorer does not rely on a database of reference spectra: instead, lipid identification routines are user defined in the declarative molecular fragmentation query language (MFQL). The software supports batch processing of multiple shotgun acquisitions by high-resolution mass mapping, precursor and neutral-loss scanning, and data-dependent MS/MS lending itself to a variety of lipidomics applications in cell biology and molecular medicine.

46 citations


Journal ArticleDOI
TL;DR: Key concepts for rare variant association for complex traits are described, some of the recent methods are surveyed, their statistical power under various scenarios are discussed, and practical guidance is provided on analyzing next‐generation sequencing data for identifying rare variants associated with complex traits.
Abstract: Although genome-wide association studies have been successful in detecting associations with common variants, there is currently an increasing interest in identifying low-frequency and rare variants associated with complex traits. Next-generation sequencing technologies make it feasible to survey the full spectrum of genetic variation in coding regions or the entire genome. The association analysis for rare variants is challenging, and traditional methods are ineffective, however, due to the low frequency of rare variants, coupled with allelic heterogeneity. Recently a battery of new statistical methods has been proposed for identifying rare variants associated with complex traits. These methods test for associations by aggregating multiple rare variants across a gene or a genomic region or among a group of variants in the genome. In this unit, we describe key concepts for rare variant association for complex traits, survey some of the recent methods, discuss their statistical power under various scenarios, and provide practical guidance on analyzing next-generation sequencing data for identifying rare variants associated with complex traits.

Journal ArticleDOI
TL;DR: This work has developed a novel method, Mutual Exclusivity Modules in Cancer (MEMo), which searches and identifies modules characterized by three properties: member genes are recurrently altered across a set of tumor samples and alteration events within the modules are mutually exclusive.
Abstract: Although individual tumors show surprisingly diverse genomic alterations, these events tend to occur in a limited number of pathways, and alterations that affect the same pathway tend to not co-occur in the same patient. While pathway analysis has been a powerful tool in cancer genomics, our knowledge of oncogenic pathway modules is incomplete. To systematically identify such modules, we have developed a novel method, Mutual Exclusivity Modules in Cancer (MEMo). The method searches and identifies modules characterized by three properties: (1) member genes are recurrently altered across a set of tumor samples; (2) member genes are known to or are likely to participate in the same biological process; and (3) alteration events within the modules are mutually exclusive. MEMo integrates multiple data types and maps genomic alterations to biological pathways. MEMo's mutual exclusivity uses a statistical model that preserves the number of alterations per gene and per sample. The MEMo software, source code and sample data sets are available for download at: http://cbio.mskcc.org/memo.

Journal ArticleDOI
TL;DR: This unit provides an introductory overview of the imputation method and describes a two‐step imputation approach that consists of the phasing of the study genotypes and the imputations of reference panel genotypes into the study haplotypes.
Abstract: Imputation is an in silico method that can increase the power of association studies by inferring missing genotypes, harmonizing data sets for meta-analyses, and increasing the overall number of markers available for association testing. This unit provides an introductory overview of the imputation method and describes a two-step imputation approach that consists of the phasing of the study genotypes and the imputation of reference panel genotypes into the study haplotypes. Detailed steps for data preparation and quality control illustrate how to run the computationally intensive two-step imputation with the high-density reference panels of the 1000 Genomes Project, which currently integrates more than 39 million variants. Additionally, the influence of reference panel selection, input marker density, and imputation settings on imputation quality are demonstrated with a simulated data set to give insight into crucial points of successful genotype imputation.

Journal ArticleDOI
TL;DR: This unit offers the details of highly sensitive and successful protocols that were initially developed largely in the lab and honed over a number of years on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre‐mRNA metabolism, or the role of noncoding RNAs.
Abstract: Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

Journal ArticleDOI
TL;DR: This unit briefly describes the two fundamentally different methods for predicting RNA structures, to find that structure with the minimum free energy of folding, as predicted by various thermodynamic parameters related to base‐pair stacking, loop lengths, and other features.
Abstract: This unit briefly describes the two fundamentally different methods for predicting RNA structures The first is to find that structure with the minimum free energy of folding, as predicted by various thermodynamic parameters related to base-pair stacking, loop lengths, and other features If one has only a single sequence, this thermodynamic approach is the best available method The second fundamental approach to RNA structure prediction is to use multiple, homologous sequences for which one can infer a common structure, and then try and predict a structure common to all of the sequences Such an approach is referred to as a comparative method or phylogenetic method of RNA structure prediction

Journal ArticleDOI
TL;DR: This module describes how to use the main features of the Discovery Environment, using bioinformatics workflows for high‐throughput sequence data as examples.
Abstract: The iPlant Collaborative is an academic consortium whose mission is to develop an informatics and social infrastructure to address the "grand challenges" in plant biology Its cyberinfrastructure supports the computational needs of the research community and facilitates solving major challenges in plant science The Discovery Environment provides a powerful and rich graphical interface to the iPlant Collaborative cyberinfrastructure by creating an accessible virtual workbench that enables all levels of expertise, ranging from students to traditional biology researchers and computational experts, to explore, analyze, and share their data By providing access to iPlant's robust data-management system and high-performance computing resources, the Discovery Environment also creates a unified space in which researchers can access scalable tools Researchers can use available Applications (Apps) to execute analyses on their data, as well as customize or integrate their own tools to better meet the specific needs of their research These Apps can also be used in workflows that automate more complicated analyses This module describes how to use the main features of the Discovery Environment, using bioinformatics workflows for high-throughput sequence data as examples

Journal ArticleDOI
TL;DR: This unit reviews the definition, classification, and management of incidental findings from genomic sequencing, with an emphasis on the key role of clinical context in defining incidental findings and determining their clinical relevance and utility.
Abstract: Genomic sequencing is becoming accurate, fast, and increasingly inexpensive, and is rapidly being incorporated into clinical practice. Incidental or secondary findings, which can occur in large numbers from genomic sequencing, are a potential barrier to the utility of this new technology due to their relatively high prevalence and the lack of evidence or guidelines available to guide their clinical interpretation. This unit reviews the definition, classification, and management of incidental findings from genomic sequencing. The unit focuses on the clinical aspects of handling incidental findings, with an emphasis on the key role of clinical context in defining incidental findings and determining their clinical relevance and utility.

Journal ArticleDOI
TL;DR: Two different synthetic strategies allowing the preparation of phosphoramidates of 6‐O‐methyl‐2‐β‐C‐methylguanosine and 2′−β‐D‐arabinouridine as model compounds for nucleosides containing two or more reactive hydroxyl groups are described.
Abstract: The ProTide (pronucleotide) approach is a prodrug strategy elaborated to deliver nucleoside monophosphate into the cell, circumventing the first and inefficient rate-limiting phosphorylation step of nucleosides and improving the cellular penetration of nucleotides. The ProTide of a nucleoside phosphate is a phosphoramidate prodrug consisting of an amino acid ester promoiety linked via P-N bond to a nucleoside aryl phosphate. Such prodrugs have increased lipophilicity and thus are capable of altering cell and tissue distribution. The ProTide technology was successfully and extensively applied to a wide variety of nucleoside phosphates, endowed with antiviral and anticancer activity. This unit describes two different synthetic strategies allowing the preparation of phosphoramidates of 6-O-methyl-2'-β-C-methylguanosine as model compounds for nucleosides having only the 5'-OH as reactive hydroxyl group, and phosphoramidates of 2'-β-D-arabinouridine (AraU) as model compounds for nucleosides containing two or more reactive hydroxyl groups.

Journal ArticleDOI
TL;DR: This protocol describes how to submit, edit, and retrieve proteomic data in Human Proteinpedia and includes information on investigators and laboratories that generated the data, as well as visualization of tandem mass spectra, stained tissue sections, protein/peptide microarrays, fluorescent micrographs, and western blots.
Abstract: Human Proteinpedia (http://www.humanproteinpedia.org) is a publicly available proteome repository for sharing human protein data derived from multiple experimental platforms. It incorporates diverse features of the human proteome including protein-protein interactions, enzyme-substrate relationships, PTMs, subcellular localization, and expression of proteins in various human tissues and cell lines in diverse biological conditions including diseases. Through a publicly distributed annotation system developed especially for proteomic data, investigators across the globe can upload, view, and edit proteomic data even before they are published. Inclusion of information on investigators and laboratories that generated the data, as well as visualization of tandem mass spectra, stained tissue sections, protein/peptide microarrays, fluorescent micrographs, and western blots, ensures quality of proteomic data assimilated in Human Proteinpedia. Many of the protein annotations submitted to Human Proteinpedia have also been made available to the scientific community through Human Protein Reference Database (http://www.hprd.org), another resource developed by our group. In this protocol, we describe how to submit, edit, and retrieve proteomic data in Human Proteinpedia. Curr. Protoc. Bioinform. 41:1.21.1-1.21.15. © 2013 by John Wiley & Sons, Inc. Keywords: mass spectrometry; tissue microarrays; biomarkers; disease proteomics; HPRD; proteotypic peptides; multiple reaction monitoring

Reference EntryDOI
TL;DR: The steps for calling CNVs from Illumina SNP array data using PennCNV and performing association analysis using R and PLINK are demonstrated.
Abstract: High-density SNP genotyping technology provides a low-cost, effective tool for conducting Genome Wide Association (GWA) studies. The wide adoption of GWA studies has indeed led to discoveries of disease- or trait-associated SNPs, some of which were subsequently shown to be causal. However, the nearly universal shortcoming of many GWA studies—missing heritability—has prompted great interest in searching for other types of genetic variation, such as copy number variation (CNV). Certain CNVs have been reported to alter disease susceptibility. Algorithms and tools have been developed to identify CNVs using SNP array hybridization intensity data. Such an approach provides an additional source of data with almost no extra cost. In this unit, we demonstrate the steps for calling CNVs from Illumina SNP array data using PennCNV and performing association analysis using R and PLINK. Curr. Protoc. Hum. Genet. 79:1.27.1-1.27.15. © 2013 by John Wiley & Sons, Inc. Keywords: copy number variations (CNV); CNV calling; genome-wide association studies; SNP genotyping array; association study; burden analysis

Journal ArticleDOI
TL;DR: A stable isotope dilution‐tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples and is useful for identifying patients with MPS types I, II, III, VI, and VII.
Abstract: Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This unit describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This new GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII.

Journal ArticleDOI
TL;DR: The PepArML meta‐search peptide identification platform for tandem mass spectra provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large‐scale searches; and an unsupervised, model‐free, machine‐learning‐based result combiner, which selects the best peptide Identification for each spectrum, estimates false‐discovery rates, and outputs pepXML format identifications.
Abstract: The PepArML meta-search peptide identification platform for tandem mass spectra provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large-scale searches; and an unsupervised, model-free, machine-learning-based result combiner, which selects the best peptide identification for each spectrum, estimates false-discovery rates, and outputs pepXML format identifications. The meta-search platform supports Mascot; Tandem with native, k-score and s-score scoring; OMSSA; MyriMatch; and InsPecT with MS-GF spectral probability scores—reformatting spectral data and constructing search configurations for each search engine on the fly. The combiner selects the best peptide identification for each spectrum based on search engine results and features that model enzymatic digestion, retention time, precursor isotope clusters, mass accuracy, and proteotypic peptide properties, requiring no prior knowledge of feature utility or weighting. The PepArML meta-search peptide identification platform often identifies two to three times more spectra than individual search engines at 10% FDR.

Journal ArticleDOI
TL;DR: This unit describes methods that can be used to quantitatively analyze the molecular crowding effects caused by cosolutes on the thermodynamic stability, hydration, and cation binding of nucleic acid structures.
Abstract: A variety of biomolecules, including nucleic acids, proteins, polysaccharides, and other soluble and insoluble low-molecular weight components, are present in living cells. These molecules occupy a significant fraction of the cellular volume (up to 40%), resulting in a highly crowded intracellular environment. This situation is referred to as molecular crowding. Although the thermodynamic stabilities of DNA structures are known to be altered in a crowded environment, less is known about the behavior of nucleic acids and their interactions with cations and water molecules under such conditions. This unit describes methods that can be used to quantitatively analyze the molecular crowding effects caused by cosolutes on the thermodynamic stability, hydration, and cation binding of nucleic acid structures.

Journal ArticleDOI
TL;DR: This unit provides an overview of the key concepts required for genetic meta-analyses, and presents strategic approaches and key decisions that must be made in the process of performing genome‐wide association study (GWAS) meta‐analyses.
Abstract: Modern genetic association studies, using genome-wide genotype data, are often underpowered. Meta-analyses of multiple studies performing genome-wide genotyping improve power and have led to the identification of thousands of genotype-trait associations. This unit provides an overview of the key concepts required for genetic meta-analyses, and presents strategic approaches and key decisions that must be made in the process of performing genome-wide association study (GWAS) meta-analyses. The commentary discusses the interpretation of GWAS meta-analysis results, complications, and some of the possible next steps once a GWAS meta-analysis has successfully identified regions associated with a trait.

Journal ArticleDOI
TL;DR: In this article, the authors describe a set of improvements that have been made to the standard Illumina protocols to make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data.
Abstract: In this unit, we describe a set of improvements that have been made to the standard Illumina protocols to make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data. Curr. Protoc. Hum. Genet. 79:18.2.1-18.2.42. © 2013 by John Wiley & Sons, Inc. Keywords: Illumina; next-generation; sequencer; protocols

Journal ArticleDOI
TL;DR: A range of different methods for the analyses of DNA methylation and hydroxymethylation at locus‐specific and genome‐wide scales is described.
Abstract: Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells DNA methylation and hydroxymethylation are epigenetic modifications found in human cells, which are deeply implicated in normal cellular processes as well as in several major human diseases Here, a range of different methods for the analyses of DNA methylation and hydroxymethylation at locus-specific and genome-wide scales is described

Journal ArticleDOI
TL;DR: This unit describes the chemical synthesis of α‐L‐threofuranosyl nucleic acid (TNA) triphosphates for thymidine, guanosine, cytidine, and the diaminopurine analog of adenosine and their incorporation into TNA oligonucleotides by enzyme‐mediated polymerization of a DNA primer‐template complex.
Abstract: This unit describes the chemical synthesis of α-l-threofuranosyl nucleic acid (TNA) triphosphates for thymidine (T), guanosine (G), cytidine (C), and the diaminopurine (D) analog of adenosine and their incorporation into TNA oligonucleotides by enzyme-mediated polymerization of a DNA primer-template complex. Starting from suitably protected threofuranosyl nucleosides, TNA triphosphates are synthesized in a single-pot reaction and purified by ion-exchange and HPLC chromatography. Purified TNA triphosphates are diluted into stock solutions and used as substrates for the synthesis of TNA oligonucleotides. Oligonucleotide synthesis is accomplished using Therminator DNA polymerase, a commercial variant of the 9oN DNA polymerase bearing the A485L mutation. Curr. Protoc. Nucleic Acid Chem. 52:4.54.1-4.54.17. © 2013 by John Wiley & Sons, Inc. Keywords: alternative nucleic acids; threose nucleic acid (TNA); triphosphates; oligonucleotide; polymerase-mediated primer extension

Journal ArticleDOI
TL;DR: This unit presents a protocol for convenient synthesis of 2′‐deoxyribo‐ and ribonucleoside 5′‐triphosphates (dNTPs and NTPs) from any natural or modified base.
Abstract: 5'-Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2'-deoxyribo- and ribonucleoside 5'-triphosphates (dNTPs and NTPs) from any natural or modified base. This one-pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non-bridging oxygen atom of the α-phosphate. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5'-triphosphates, as well as analysis and confirmation of natural and sulfur- or selenium-modified nucleic acids, are described in this protocol unit.

Journal ArticleDOI
TL;DR: This unit describes cloning strategies, procedures to turn the intended design into virus, and quality analyses of resultant adenoviral vectors for intensive use as an anticancer agent.
Abstract: Oncolytic adenoviruses are designed to take advantage of the virus' native ability to replicate in cancer cells to induce oncolysis. Subsequently, the released viral progeny spread and kill the neighboring cancer cells. These characteristics, together with the ability of adenovirus to infect a broad spectrum of cells, its well understood replication machinery, and relative ease of manufacture have led to the intensive use of adenovirus as an anticancer agent. This unit describes cloning strategies, procedures to turn the intended design into virus, and quality analyses of resultant adenoviral vectors. Most of these procedures were optimized especially for oncolytic adenoviral vectors. Curr. Protoc. Hum. Genet. 78:12.14.1-12.14.21. © 2013 by John Wiley & Sons, Inc. Keywords: adenovirus; oncolytic; production; design; cloning; strategy; amplification; purification

Journal ArticleDOI
TL;DR: The challenges in patient selection and consent, and the technical issues surrounding test interpretation and reporting are highlighted, and practical solutions that allow for genome‐wide sequencing to be implemented in current clinical practice are provided.
Abstract: The recent entry of "whole" exome/"whole" genome sequencing into limited clinical practice has led to a progression of the availability of genome-scale testing beyond deletion/duplication copy number arrays. This unit provides a considered approach to the implementation of such testing in routine clinical practice. Specifically, we will highlight the challenges in patient selection and consent, and the technical issues surrounding test interpretation and reporting. The unit will then provide practical solutions that allow for genome-wide sequencing to be implemented in current clinical practice.