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Nature Reviews. Genetics Jan 2014Advances in next-generation sequencing (NGS) technologies have rapidly improved sequencing fidelity and substantially decreased sequencing error rates. However, given... (Review)
Review
Advances in next-generation sequencing (NGS) technologies have rapidly improved sequencing fidelity and substantially decreased sequencing error rates. However, given that there are billions of nucleotides in a human genome, even low experimental error rates yield many errors in variant calls. Erroneous variants can mimic true somatic and rare variants, thus requiring costly confirmatory experiments to minimize the number of false positives. Here, we discuss sources of experimental errors in NGS and how replicates can be used to abate such errors.
Topics: Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Research Design; Sample Size
PubMed: 24322726
DOI: 10.1038/nrg3655 -
Cell Sep 2013Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome... (Review)
Review
Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome characterization over sequencing's 40-year history, the scope of inquiry has commensurately broadened. Massively parallel sequencing has proven revolutionary, shifting the paradigm of genomics to address biological questions at a genome-wide scale. Sequencing now empowers clinical diagnostics and other aspects of medical care, including disease risk, therapeutic identification, and prenatal testing. This Review explores the current state of genomics in the massively parallel sequencing era.
Topics: Animals; Disease; Genetic Variation; Genomics; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Humans; Mutation; Sequence Analysis, DNA
PubMed: 24074859
DOI: 10.1016/j.cell.2013.09.006 -
A survey of the approaches for identifying differential methylation using bisulfite sequencing data.Briefings in Bioinformatics Sep 2018DNA methylation is an important epigenetic mechanism that plays a crucial role in cellular regulatory systems. Recent advancements in sequencing technologies now enable... (Review)
Review
DNA methylation is an important epigenetic mechanism that plays a crucial role in cellular regulatory systems. Recent advancements in sequencing technologies now enable us to generate high-throughput methylation data and to measure methylation up to single-base resolution. This wealth of data does not come without challenges, and one of the key challenges in DNA methylation studies is to identify the significant differences in the methylation levels of the base pairs across distinct biological conditions. Several computational methods have been developed to identify differential methylation using bisulfite sequencing data; however, there is no clear consensus among existing approaches. A comprehensive survey of these approaches would be of great benefit to potential users and researchers to get a complete picture of the available resources. In this article, we present a detailed survey of 22 such approaches focusing on their underlying statistical models, primary features, key advantages and major limitations. Importantly, the intrinsic drawbacks of the approaches pointed out in this survey could potentially be addressed by future research.
Topics: Computational Biology; CpG Islands; DNA Methylation; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Humans; Logistic Models; Markov Chains; Sequence Analysis, DNA; Sulfites
PubMed: 28334228
DOI: 10.1093/bib/bbx013 -
Journal of Biomolecular Techniques : JBT Sep 2019The development and expansion of the core facility concept are <4 decades old. The factors that favored the use of shared instrumentation facilities and the requirement...
The development and expansion of the core facility concept are <4 decades old. The factors that favored the use of shared instrumentation facilities and the requirement for expert staff are covered by one of the founders of the Association of Biomolecular Resource Facilities (ABRF). During the decade when grants for shared instruments and the release of modern, automated instruments flourished, protocol development for those new instruments came primarily out of laboratories of the type we now call core facilities. Because of the new technologies available, new protocols were required to meet the needs of research communities, and much of the development took place in the diverse core facilities. Furthermore, technology development itself was a frequent activity in core facilities. Although guidelines for the operation of core facilities were not available in the early days of core facility operation, they evolved over time. Cost recovery was, and is still, one of the most problematic issues for core facilities. ABRF-supported research groups offered members opportunities to evaluate their capabilities with both lab-developed protocols and study-specified protocols and with comparative data collected in surveys of core facilities. Research groups are a premier activity of ABRF and its members. More new developing technologies have followed using this pattern of collaboration among core facilities and with industry. The exhibition floor at ABRF annual meetings shows off many of the results of these collaborations.
Topics: Equipment and Supplies; High-Throughput Nucleotide Sequencing; History, 20th Century; Humans; Laboratories; Technology
PubMed: 31452646
DOI: 10.7171/jbt.19-3003-002 -
PloS One 2015The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are...
The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications.
Topics: DNA; Genome, Human; Genotyping Techniques; High-Throughput Nucleotide Sequencing; Humans; INDEL Mutation; Polymorphism, Single Nucleotide; Reproducibility of Results
PubMed: 26382624
DOI: 10.1371/journal.pone.0138259 -
Clinical Microbiology and Infection :... Jul 2012A metamobilome is defined as a metagenome of circular genetic elements within a certain community. Metagenomic analyses of plasmids provide insights into the composition... (Review)
Review
A metamobilome is defined as a metagenome of circular genetic elements within a certain community. Metagenomic analyses of plasmids provide insights into the composition and structure of environmental plasmid communities. It is a promising method that will provide information about the types of plasmids that are present within environmental samples, and will give overviews about occurrences of plasmids as well as accessory genetic elements carried on these plasmids. A metamobilome library was constructed by combining multiple displacement amplification with pyrosequencing. This method provided a fast, efficient and unbiased strategy to investigate the communal gene pool of circular genetic elements (the metamobilome). We compared our wastewater metamobilome library with a wastewater metagenome library, against chromosomes, plasmids, phages and IS element databases, respectively. This showed that very few strictly chromosomal reads were present in our metamobilome library. Furthermore, data analysis showed that our library was strongly enriched for genes encoding plasmid-selfish traits, such as stability and conjugation, and most strikingly several hundred new putative plasmid replicases have been recovered.
Topics: DNA, Circular; High-Throughput Nucleotide Sequencing; Humans; Interspersed Repetitive Sequences; Metagenomics; Plasmids; Sewage
PubMed: 22647039
DOI: 10.1111/j.1469-0691.2012.03862.x -
Chinese Journal of Cancer Oct 2012With the development and improvement of new sequencing technology, next-generation sequencing (NGS) has been applied increasingly in cancer genomics research over the... (Review)
Review
With the development and improvement of new sequencing technology, next-generation sequencing (NGS) has been applied increasingly in cancer genomics research over the past decade. More recently, NGS has been adopted in clinical oncology to advance personalized treatment of cancer. NGS is used to identify novel and rare cancer mutations, detect familial cancer mutation carriers, and provide molecular rationale for appropriate targeted therapy. Compared to traditional sequencing, NGS holds many advantages, such as the ability to fully sequence all types of mutations for a large number of genes (hundreds to thousands) in a single test at a relatively low cost. However, significant challenges, particularly with respect to the requirement for simpler assays, more flexible throughput, shorter turnaround time, and most importantly, easier data analysis and interpretation, will have to be overcome to translate NGS to the bedside of cancer patients. Overall, continuous dedication to apply NGS in clinical oncology practice will enable us to be one step closer to personalized medicine.
Topics: High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Precision Medicine; Sequence Analysis, DNA
PubMed: 22980418
DOI: 10.5732/cjc.012.10216 -
JAMA Mar 2019
Topics: Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30763433
DOI: 10.1001/jama.2018.21669 -
International Journal of Legal Medicine Jul 2020In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its... (Review)
Review
In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its introduction to the field of forensics was slower, mainly due to lack of accredited sequencers, kits, and relatively higher sequencing error rates as compared with standardized Sanger sequencing. Currently, a majority of the problematic issues have been solved, which is proven by the body of reports in the literature. Here, we discuss the utility of NGS sequencing in forensics, emphasizing the advantages, issues, the technical aspects of the experiments, commercial solutions, and the potentially interesting applications of MPS.
Topics: Forensic Sciences; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 32451905
DOI: 10.1007/s00414-020-02294-0 -
Genome Biology Jul 2023Although long-read RNA-seq is increasingly applied to characterize full-length transcripts it can also enable detection of nucleotide variants, such as genetic mutations...
Although long-read RNA-seq is increasingly applied to characterize full-length transcripts it can also enable detection of nucleotide variants, such as genetic mutations or RNA editing sites, which is significantly under-explored. Here, we present an in-depth study to detect and analyze RNA editing sites in long-read RNA-seq. Our new method, L-GIREMI, effectively handles sequencing errors and read biases. Applied to PacBio RNA-seq data, L-GIREMI affords a high accuracy in RNA editing identification. Additionally, our analysis uncovered novel insights about RNA editing occurrences in single molecules and double-stranded RNA structures. L-GIREMI provides a valuable means to study nucleotide variants in long-read RNA-seq.
Topics: RNA-Seq; Transcriptome; RNA Editing; Nucleotides; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing
PubMed: 37474948
DOI: 10.1186/s13059-023-03012-w