-
International Journal of Legal Medicine Jan 2022Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful...
Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful validation and establishment of several pyrosequencing assays using a PyroMark® Q48 Autoprep instrument (Qiagen, Hilden, Germany), we decided to expand the method further purchasing a second instrument. But after initializing this second instrument side by side with the first, the majority of analyses failed (97 samples of 133 samples (73%)). The number of error messages increased rapidly and the average RFU values decreased. After purchasing two anti-vibration weighing tables for the PyroMark® instruments and repeating the analyses under the same conditions and with identical samples the results improved considerably, 115 samples of 130 samples (88%) showed successful and reproducible results. These findings demonstrate the impact of vibrations and percussions on PyroMark® Q48 Autoprep performance and the reliability of methylation analyses.
Topics: CpG Islands; DNA Methylation; High-Throughput Nucleotide Sequencing; Humans; Reproducibility of Results; Sequence Analysis, DNA; Vibration
PubMed: 34637025
DOI: 10.1007/s00414-021-02716-7 -
Annual Review of Genomics and Human... Aug 2016The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce... (Review)
Review
The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce data at or beyond the genome scale. Since the introduction of these technologies, the number of applications and methods that leverage the power of genome-scale sequencing has increased at an exponential pace. This review highlights recent concepts, technologies, and methods from next-generation sequencing to illustrate the breadth and depth of the applications and research areas that are driving progress in genomics.
Topics: Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 27362342
DOI: 10.1146/annurev-genom-083115-022413 -
PLoS Computational Biology Mar 2020Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a...
Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a parallel manner, the number of core facilities that provide bioinformatics support are also increasing. Notably, the generation of complex large datasets has necessitated the development of bioinformatics support core facilities that aid laboratory scientists with cost-effective and efficient data management, analysis, and interpretation. In this article, we address the challenges-related to communication, good laboratory practice, and data handling-that may be encountered in core support facilities when providing bioinformatics support, drawing on our own experiences working as support bioinformaticians on multidisciplinary research projects. Most importantly, the article proposes a list of guidelines that outline how these challenges can be preemptively avoided and effectively managed to increase the value of outputs to the end user, covering the entire research project lifecycle, including experimental design, data analysis, and management (i.e., sharing and storage). In addition, we highlight the importance of clear and transparent communication, comprehensive preparation, appropriate handling of samples and data using monitoring systems, and the employment of appropriate tools and standard operating procedures to provide effective bioinformatics support.
Topics: Biomedical Research; Communication; Computational Biology; High-Throughput Nucleotide Sequencing; Humans; Research Design
PubMed: 32214318
DOI: 10.1371/journal.pcbi.1007531 -
Mitochondrion Jul 2014Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels.... (Review)
Review
Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16,569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control.
Topics: DNA Copy Number Variations; DNA, Mitochondrial; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Mitochondrial Diseases; Polymorphism, Single Nucleotide
PubMed: 24859348
DOI: 10.1016/j.mito.2014.05.004 -
Genomics Dec 2014We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by... (Review)
Review
We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by microbiologists and biochemists, who used experimental evolutionary change as a tool to understand structure-function relationships. These studies attracted the interest of evolutionary biologists who recognized the power of the approach to address issues such as the tempo of adaptive change, the costs and benefits of sex, parallelism, and the role which contingency plays in the evolutionary process. In the 1980s and 1990s, an ever-expanding body of microbial, physiological and biochemical data, together with new technologies for manipulating microbial genomes, allowed such questions to be addressed in ever-increasing detail. Since then, technological advances leading to low-cost, high-throughput DNA sequencing have made it possible for these and other fundamental questions in evolutionary biology to be addressed at the molecular level.
Topics: Databases, Genetic; Directed Molecular Evolution; Evolution, Molecular; Genome, Microbial; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century
PubMed: 25315137
DOI: 10.1016/j.ygeno.2014.10.004 -
Journal of Biomolecular Techniques : JBT Dec 2020Advances in next-generation sequencing technologies have allowed RNA sequencing to become an increasingly time efficient, cost-effective, and accessible tool for genomic...
Advances in next-generation sequencing technologies have allowed RNA sequencing to become an increasingly time efficient, cost-effective, and accessible tool for genomic research. We present here an automated and miniaturized workflow for RNA library preparation that minimizes reagent usage and processing time required per sample to generate Illumina compatible libraries for sequencing. The reduced-volume libraries show similar behavior to full-scale libraries with comparable numbers of genes detected and reproducible clustering of samples.
Topics: Automation; Gene Library; Genomics; High-Throughput Nucleotide Sequencing; RNA; RNA-Seq; Reproducibility of Results; Workflow
PubMed: 33100919
DOI: 10.7171/jbt.20-3104-004 -
Analytical and Bioanalytical Chemistry Jun 2012The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the... (Review)
Review
The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.
Topics: Animals; DNA; Emulsions; Equipment Design; High-Throughput Nucleotide Sequencing; Humans; Microfluidics; Polymerase Chain Reaction
PubMed: 22451171
DOI: 10.1007/s00216-012-5914-x -
Circulation Research Sep 2015
Topics: Cost-Benefit Analysis; Heart Diseases; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26358106
DOI: 10.1161/CIRCRESAHA.115.307344 -
Methods (San Diego, Calif.) May 2019A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such... (Review)
Review
A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.
Topics: Animals; Genetic Engineering; High-Throughput Nucleotide Sequencing; Humans; Mutation; RNA, Catalytic
PubMed: 30738128
DOI: 10.1016/j.ymeth.2019.02.001 -
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