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Journal of Visualized Experiments : JoVE Feb 2023Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in...
Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.
Topics: Genome, Mitochondrial; Polymorphism, Single Nucleotide; Genotype; Sequence Analysis, DNA; DNA, Mitochondrial; High-Throughput Nucleotide Sequencing
PubMed: 36847373
DOI: 10.3791/64361 -
Philosophical Transactions of the Royal... Jan 2015The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear... (Review)
Review
The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past.
Topics: Animals; DNA; Genomics; High-Throughput Nucleotide Sequencing; History, Ancient; Humans
PubMed: 25487338
DOI: 10.1098/rstb.2013.0387 -
Methods in Molecular Biology (Clifton,... 2013Despite the influx of high throughput sequencing techniques, there is still a niche for low-medium throughput genotyping technologies for small-scale screening and...
Despite the influx of high throughput sequencing techniques, there is still a niche for low-medium throughput genotyping technologies for small-scale screening and validation purposes. Pyrosequencing is a genotyping assay based on sequencing-by-synthesis. Short runs of sequence around each polymorphism are generated, allowing for internal controls for each sample. Pyrosequencing can also be utilized to identify tri-allelic, indel, and short repeat polymorphisms, as well as determining allele percentages for methylation or pooled sample assessment. This range of applications makes it well-suited to the research laboratory as a one-stop system.
Topics: Alleles; Genotyping Techniques; High-Throughput Nucleotide Sequencing; Humans; INDEL Mutation; Polymorphism, Genetic
PubMed: 23824851
DOI: 10.1007/978-1-62703-435-7_6 -
Journal of Agricultural and Food... Jan 2014The increased consumption of fish products, as well as the occurrence of exotic fish species in the Mediterranean Sea and in the fish market, has increased the risk of...
The increased consumption of fish products, as well as the occurrence of exotic fish species in the Mediterranean Sea and in the fish market, has increased the risk of commercial fraud. Furthermore, the great amount of processed seafood products has greatly limited the application of classic identification systems. DNA-based identification allows a clear and unambiguous detection of polymorphisms between species, permitting differentiation and identification of both commercial fraud and introduction of species with potential toxic effects on humans. In this study, a novel DNA-based approach for differentiation of fish species based on pyrosequencing technology has been developed. Raw and processed fish products were tested, and up to 25 species of fish belonging to Clupeiformes and Pleuronectiformes groups were uniquely and rapidly identified. The proper identification based on short and unique genetic sequence signatures demonstrates that this approach is promising and cost-effective for large-scale surveys.
Topics: Animals; Fishes; Food Contamination; High-Throughput Nucleotide Sequencing; Seafood; Species Specificity
PubMed: 24350776
DOI: 10.1021/jf403545m -
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 -
Nature Biotechnology Jan 2012
Topics: Drug Industry; Genes, Neoplasm; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Medical Oncology; Neoplasms
PubMed: 22231083
DOI: 10.1038/nbt0112-14 -
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 -
Methods in Molecular Biology (Clifton,... 2019As in any endeavor, the strategy applied to a genome project can mean the difference between success and failure. This is especially important when limited funding often... (Review)
Review
As in any endeavor, the strategy applied to a genome project can mean the difference between success and failure. This is especially important when limited funding often means only a single approach may be tried at a given time. Although the advance of all areas of genomics and transcriptomics in recent years has led to an embarrassment of riches, methods in the field have not quite reached the turn-key production status for all species, despite being closer than ever. Here I contrast and compare the technical approaches to genome projects in the hope of enabling strategy choices with higher probabilities of success. Finally, I review the new technologies that are not yet widely distributed which are revolutionizing the future of genomics.
Topics: Animals; Arthropods; Genome, Insect; Genomics; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 30414106
DOI: 10.1007/978-1-4939-8775-7_1 -
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 -
Molecular Medicine Reports Feb 2022The present study aimed to develop a reliable pyrosequencing method to detect four single nucleotide polymorphisms (SNPs) of the flavin‑containing monooxygenase 3 ()...
The present study aimed to develop a reliable pyrosequencing method to detect four single nucleotide polymorphisms (SNPs) of the flavin‑containing monooxygenase 3 () gene and to compare the ethnic differences in their allelic frequencies. The pyrosequencing method was used to detect four SNPs, namely, c.855C>T (N285N, rs909530), c.441C>T (S147S, rs1800822), c.923A>G (E308G, rs2266780) and c.472G>A (E158K, rs2266782). The allelic frequencies of these SNPs in 122 unrelated Korean subjects were as follows: i) 44.7% for c.855C>T; ii) 23.4% for c.441C>T; iii) 23.0% for c.923A>G; and iv) 27.1% for c.472G>A. Linkage disequilibrium (LD) analysis revealed that the SNPs c.923A>G and c.472G>A exhibited a strong LD (D'=0.8289, r=0.5332). In conclusion, the pyrosequencing method developed in this study was successfully applied to detect the c.855C>T, c.441C>T, c.923A>G and c.472G>A SNPs of .
Topics: Adult; Asian People; Gene Frequency; Healthy Volunteers; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Oxygenases; Polymorphism, Single Nucleotide; Reproducibility of Results; Republic of Korea; Young Adult
PubMed: 34913068
DOI: 10.3892/mmr.2021.12564