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The Veterinary Clinics of North... Aug 2020The sequencing and assembly of a reference genome for the horse has been revolutionary for investigation of horse health and performance. Next-generation sequencing... (Review)
Review
The sequencing and assembly of a reference genome for the horse has been revolutionary for investigation of horse health and performance. Next-generation sequencing (NGS) methods represent a second revolution in equine genomics. Researchers can align and compare DNA and RNA sequencing data to the reference genome to explore variation that may contribute or be attributed to disease. NGS has also facilitated the translation of research discovery to clinically relevant applications. This article discusses the history and development of NGS, details some of the available sequencing platforms, and describes currently available applications in the context of both discovery and clinical settings.
Topics: Animals; Genomics; High-Throughput Nucleotide Sequencing; Horse Diseases; Horses; Sequence Analysis, DNA
PubMed: 32654781
DOI: 10.1016/j.cveq.2020.03.002 -
Genes Jan 2019The adoption of single molecule real-time (SMRT) sequencing [...].
The adoption of single molecule real-time (SMRT) sequencing [...].
Topics: Animals; High-Throughput Nucleotide Sequencing; Humans; Plants; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 30621217
DOI: 10.3390/genes10010024 -
Electrophoresis Jan 2023The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science.... (Review)
Review
The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.
Topics: Forensic Medicine; Genomics; Forensic Sciences; High-Throughput Nucleotide Sequencing; Forensic Genetics
PubMed: 36168852
DOI: 10.1002/elps.202200177 -
Nature Genetics Nov 2022
Topics: RNA-Seq; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing; Gene Expression Profiling
PubMed: 36333507
DOI: 10.1038/s41588-022-01217-6 -
Methods in Molecular Biology (Clifton,... 2023Pyrosequencing is a DNA sequencing-by-synthesis technique that can quantitatively detect single-nucleotide polymorphisms (SNPs). With pyrosequencing, the level of DNA...
Pyrosequencing is a DNA sequencing-by-synthesis technique that can quantitatively detect single-nucleotide polymorphisms (SNPs). With pyrosequencing, the level of DNA methylation can be calculated according to the ratio of artificial cytosine/thymine SNPs produced by bisulfite conversion at each CpG site. This analysis method provides a reproducible and accurate measurement of methylation levels at CpG sites near sequencing primers with high quantitative resolution. DNA methylation plays an important role in mammalian development and cellular physiology; alterations in DNA methylation patterns have been implicated in several common diseases as well as cancers and imprinting disorders. Evaluating DNA methylation levels via pyrosequencing is useful for identifying biomarkers that could help with the diagnosis, prognosis, treatment selection, and onset risk assessment for several diseases. We describe the principles of pyrosequencing and detail a bisulfite pyrosequencing protocol based on our experience and the PyroMark Q24 User Manual.
Topics: Animals; CpG Islands; Cytosine; DNA Methylation; DNA Primers; High-Throughput Nucleotide Sequencing; Mammals; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Sulfites; Thymine
PubMed: 36173562
DOI: 10.1007/978-1-0716-2724-2_1 -
GigaScience Aug 2018With the rapid development of next-generation sequencing technology, ever-increasing quantities of genomic data pose a tremendous challenge to data processing.... (Review)
Review
With the rapid development of next-generation sequencing technology, ever-increasing quantities of genomic data pose a tremendous challenge to data processing. Therefore, there is an urgent need for highly scalable and powerful computational systems. Among the state-of-the-art parallel computing platforms, Apache Spark is a fast, general-purpose, in-memory, iterative computing framework for large-scale data processing that ensures high fault tolerance and high scalability by introducing the resilient distributed dataset abstraction. In terms of performance, Spark can be up to 100 times faster in terms of memory access and 10 times faster in terms of disk access than Hadoop. Moreover, it provides advanced application programming interfaces in Java, Scala, Python, and R. It also supports some advanced components, including Spark SQL for structured data processing, MLlib for machine learning, GraphX for computing graphs, and Spark Streaming for stream computing. We surveyed Spark-based applications used in next-generation sequencing and other biological domains, such as epigenetics, phylogeny, and drug discovery. The results of this survey are used to provide a comprehensive guideline allowing bioinformatics researchers to apply Spark in their own fields.
Topics: Animals; Computational Biology; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mice; Software
PubMed: 30101283
DOI: 10.1093/gigascience/giy098 -
Clinical Chemistry Jan 2020The newest advances in DNA sequencing are based on technologies that perform massively parallel sequencing (MPS). Since 2006, the output from MPS platforms has increased... (Review)
Review
BACKGROUND
The newest advances in DNA sequencing are based on technologies that perform massively parallel sequencing (MPS). Since 2006, the output from MPS platforms has increased from 20 Mb to >7 Tb. First-generation MPS platforms amplify individual DNA molecules to multiple copies and then interrogate the sequence of those molecules. Second-generation MPS analyzes single unamplified molecules to generate much longer sequence reads but with less output than first-generation MPS and lower first-pass accuracy. With MPS technologies, it is now possible to analyze genomes, exomes, a defined subset of genes, transcriptomes, and even methylation across the genome. These technologies have and will continue to completely transform the clinical practice.
CONTENT
The major first- and second-generation MPS platforms and how they are used in clinical practice are discussed.
SUMMARY
The ability to sequence terabases of DNA per run on an MPS platform will dramatically change how DNA sequencing is used in clinical practice. Currently, MPS of targeted gene panels is the most common use of this technology clinically, but as the cost for genome sequencing inches downward to $100, this may soon become the method of choice (with the caveat that, at least in the near term, clinical-grade genome sequencing with interpretation may cost much more than $100). Other uses of this technology include sequencing of a mixture of bacterial and viral species (metagenomics), as well as the characterization of methylation across the genome.
Topics: DNA; DNA Methylation; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Nanostructures; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 31811004
DOI: 10.1373/clinchem.2019.303305 -
The Veterinary Clinics of North... Mar 2023Next-generation sequencing (NGS) was initially developed to aid sequencing of the human genome. This molecular method is cost effective for sequencing and characterizing... (Review)
Review
Next-generation sequencing (NGS) was initially developed to aid sequencing of the human genome. This molecular method is cost effective for sequencing and characterizing genomes, not only those of humans or animals but also those of bacteria and other pathogens. However, rather than sequencing a single organism, a targeted NGS method can be used to specifically amplify pathogens of interest in a clinical sample for detection and characterization by sequencing. Targeted NGS is an ideal method for ruminant syndromic testing due to its ability to detect a variety of pathogens in a sample with a single test.
Topics: Humans; Animals; Bacteria; High-Throughput Nucleotide Sequencing
PubMed: 36731996
DOI: 10.1016/j.cvfa.2022.09.003 -
Methods in Molecular Biology (Clifton,... 2017A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful...
A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful approach for discovering the causal genes underlying phenotypes. For genetic mapping, the process of genotyping was previously a major rate-limiting step. Modern sequencing technology has greatly improved the resolution and speed of genetic mapping by reducing the time, labor, and cost per genotyping marker. In addition, the ability to perform genotyping-by-sequencing (GBS) has facilitated large-scale population genetic analyses by providing a simpler way to survey segregating genetic variation in natural populations. Here we present two protocols for GBS, using the Illumina platform, that can be applied to a wide range of genotyping projects in different species. The first protocol is for genotyping a subset of marker positions genome-wide using restriction digestion, and the second is for preparing inexpensive paired-end whole-genome libraries. We discuss the suitability of each approach for different genotyping applications and provide notes for adapting these protocols for use with a liquid-handling robot.
Topics: Electrophoresis, Agar Gel; Genotype; High-Throughput Nucleotide Sequencing
PubMed: 27822868
DOI: 10.1007/978-1-4939-6442-0_16 -
Advances in Experimental Medicine and... 2016The field of proteogenomics has been driven by combined advances in next-generation sequencing (NGS) and proteomic methods. NGS technologies are now both rapid and... (Review)
Review
The field of proteogenomics has been driven by combined advances in next-generation sequencing (NGS) and proteomic methods. NGS technologies are now both rapid and affordable, making it feasible to include sequencing in the clinic and academic research setting. Alongside the improvements in sequencing technologies, methods in high throughput proteomics have increased the depth of coverage and the speed of analysis. The integration of these data types using continuously evolving bioinformatics methods allows for improvements in gene and protein annotation, and a more comprehensive understanding of biological systems.
Topics: Alternative Splicing; Amino Acid Sequence; Amino Acid Substitution; Base Sequence; High-Throughput Nucleotide Sequencing; Humans; Molecular Sequence Annotation; Polymorphism, Single Nucleotide; Proteogenomics; Proteome; Sequence Alignment; Software; Time Factors
PubMed: 27686803
DOI: 10.1007/978-3-319-42316-6_2