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Methods in Molecular Biology (Clifton,... 2011Single nucleotide polymorphisms (SNPs) are single base differences between haplotypes. SNPs are abundant in many species and valuable as markers for genetic map...
Single nucleotide polymorphisms (SNPs) are single base differences between haplotypes. SNPs are abundant in many species and valuable as markers for genetic map construction, modern molecular breeding programs, and quantitative genetic studies. SNPs are readily mined from genomic DNA or cDNA sequence obtained from individuals having two or more distinct genotypes. While automated Sanger sequencing has become less expensive over time, it is still costly to acquire deep Sanger sequence from several genotypes. "Next-generation" DNA sequencing technologies that utilize new chemistries and massively parallel approaches have enabled DNA sequences to be acquired at extremely high depths of coverage faster and for less cost than traditional sequencing. One such method is represented by the Roche/454 Life Sciences GS-FLX Titanium Series, which currently uses pyrosequencing to produce up to 400-600 million bases of DNA sequence/run (>1 million reads, ~400 bp/read). This chapter discusses the use of high-throughput pyrosequencing for SNP discovery by focusing on 454 sequencing of maize cDNA, the development of a computational pipeline for polymorphism detection, and the subsequent identification of over 7,000 putative SNPs between Mo17 and B73 maize. In addition, alternative alignment and polymorphism detection strategies that implement Illumina short reads, data processing and visualization tools, and reduced representation techniques that reduce the sequencing of repeat DNA, thus enabling efficient analysis of genome sequence, are discussed.
Topics: Amino Acid Sequence; Base Sequence; Chromosome Mapping; Computational Biology; DNA, Plant; Expressed Sequence Tags; Gene Expression Profiling; Genome, Plant; Genotype; Haplotypes; High-Throughput Nucleotide Sequencing; Molecular Sequence Data; Polymorphism, Single Nucleotide; Zea mays
PubMed: 21365494
DOI: 10.1007/978-1-61779-065-2_15 -
Circulation Research Sep 2015
Topics: Cost-Benefit Analysis; Heart Diseases; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26358106
DOI: 10.1161/CIRCRESAHA.115.307344 -
Clinical Microbiology and Infection :... Jan 2013Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives... (Review)
Review
Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.
Topics: High-Throughput Nucleotide Sequencing; Humans; Metagenome; Molecular Diagnostic Techniques; Virology
PubMed: 23279287
DOI: 10.1111/1469-0691.12056 -
Topics in Current Chemistry 2014High-throughput next generation sequencing (NGS) has been quickly adapted into many aspects of biomedical research and begun to engage with the clinical practice. The... (Review)
Review
High-throughput next generation sequencing (NGS) has been quickly adapted into many aspects of biomedical research and begun to engage with the clinical practice. The latter aspect will enable the application of genomic knowledge into clinical practice in this and next decades and will profoundly change the diagnosis, prognosis and treatment of many human diseases. It will further demand both philosophical and medical curriculum reforms in the training of our future physicians. However, significant huddles need to be overcome before an ultimate application of NGS in genomic medicine can be practical and fruitful.
Topics: High-Throughput Nucleotide Sequencing; Humans
PubMed: 22648865
DOI: 10.1007/128_2012_329 -
Genome Biology Jun 2013The sequencing of large and complex genomes of crop species, facilitated by new sequencing technologies and bioinformatic approaches, has provided new opportunities for... (Review)
Review
The sequencing of large and complex genomes of crop species, facilitated by new sequencing technologies and bioinformatic approaches, has provided new opportunities for crop improvement. Current challenges include understanding how genetic variation translates into phenotypic performance in the field.
Topics: Breeding; Crops, Agricultural; Genetic Variation; Genome, Plant; Genomics; High-Throughput Nucleotide Sequencing; History, 21st Century; Phenotype
PubMed: 23796126
DOI: 10.1186/gb-2013-14-6-206 -
Methods in Molecular Biology (Clifton,... 2019Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture...
Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are coconfined through a microfluidic device within the droplets where they undergo cell lysis and RNA hybridiztion. After breaking the droplets and pooling the hybridized particles, reverse transcription, PCR, and sequencing in single reactions allow to generate data from thousands of single-cell transcriptomes while maintaining information on the cellular origin of each transcript.
Topics: Animals; Equipment Design; Gene Expression Profiling; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Lab-On-A-Chip Devices; Single-Cell Analysis; Transcriptome
PubMed: 31028633
DOI: 10.1007/978-1-4939-9240-9_6 -
Current Opinion in Microbiology Oct 2012
Topics: Biota; Ecosystem; Genome, Bacterial; High-Throughput Nucleotide Sequencing
PubMed: 23084564
DOI: 10.1016/j.mib.2012.10.001 -
Molecular Ecology Apr 2012Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific... (Review)
Review
Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.
Topics: DNA; Environmental Monitoring; High-Throughput Nucleotide Sequencing; Metagenomics
PubMed: 22486820
DOI: 10.1111/j.1365-294X.2012.05538.x -
Revue Scientifique Et Technique... Apr 2016Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become... (Review)
Review
Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique.
Topics: Animals; Genome, Viral; High-Throughput Nucleotide Sequencing; Molecular Epidemiology; Terminology as Topic; Virus Diseases; Viruses
PubMed: 27217167
DOI: 10.20506/rst.35.1.2416 -
Expert Review of Molecular Diagnostics Sep 2016The last two decades have witnessed revolutionary changes in clinical diagnostics, fueled by the Human Genome Project and advances in high throughput, Next Generation... (Review)
Review
INTRODUCTION
The last two decades have witnessed revolutionary changes in clinical diagnostics, fueled by the Human Genome Project and advances in high throughput, Next Generation Sequencing (NGS). We review the current state of sequencing-based pediatric diagnostics, associated challenges, and future prospects.
AREAS COVERED
We present an overview of genetic disease in children, review the technical aspects of Next Generation Sequencing and the strategies to make molecular diagnoses for children with genetic disease. We discuss the challenges of genomic sequencing including incomplete current knowledge of variants, lack of data about certain genomic regions, mosaicism, and the presence of regions with high homology. Expert commentary: NGS has been a transformative technology and the gap between the research and clinical communities has never been so narrow. Therapeutic interventions are emerging based on genomic findings and the applications of NGS are progressing to prenatal genetics, epigenomics and transcriptomics.
Topics: Adolescent; Child; Child, Preschool; Female; Genetic Diseases, Inborn; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Male; Mosaicism
PubMed: 27388938
DOI: 10.1080/14737159.2016.1209411