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Genomics, Proteomics & Bioinformatics Oct 2016The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology,... (Review)
Review
The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assembly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.
Topics: Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Nanopores; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA
PubMed: 27646134
DOI: 10.1016/j.gpb.2016.05.004 -
Methods in Molecular Biology (Clifton,... 2019DNA methylation is a process by which methyl groups are added to cytosine or adenine. DNA methylation can change the activity of the DNA molecule without changing the...
DNA methylation is a process by which methyl groups are added to cytosine or adenine. DNA methylation can change the activity of the DNA molecule without changing the sequence. Methylation of 5-methylcytosine (5mC) is widespread in both eukaryotes and prokaryotes, and it is a very important epigenetic modification event, which can regulate gene activity and influence a number of key processes such as genomic imprinting, cell differentiation, transcriptional regulation, and chromatin remodeling. Profiling DNA methylation across the genome is critical to understanding the influence of methylation in normal biology and diseases including cancer. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC), and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the methylation regulation. Genome-wide analyses such as microarrays and next-generation sequencing technologies have been used to assess large fractions of the methylome. A number of different quantitative approaches have also been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing, pyrosequencing etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this chapter is to provide the methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.
Topics: 5-Methylcytosine; Animals; DNA; DNA Methylation; Epigenesis, Genetic; Epigenomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30547463
DOI: 10.1007/978-1-4939-8916-4_12 -
European Journal of Human Genetics :... Jan 2016We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS)...
We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a 'rating system' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.
Topics: Accreditation; Biomarkers; Databases, Genetic; Europe; Gene Expression; Genetic Diseases, Inborn; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Incidental Findings; Information Dissemination; Informed Consent; Proteins; Research Design; Sensitivity and Specificity
PubMed: 26508566
DOI: 10.1038/ejhg.2015.226 -
Biomolecules Jul 2021Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is... (Review)
Review
Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.
Topics: Animals; Base Sequence; DNA; Genome; Genomics; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Humans; RNA, Messenger; Sequence Analysis, DNA; Sequence Analysis, RNA; Whole Genome Sequencing
PubMed: 34439777
DOI: 10.3390/biom11081111 -
Journal of Clinical Microbiology Dec 2019Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so... (Review)
Review
Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.
Topics: Clinical Laboratory Services; Clinical Laboratory Techniques; Communicable Diseases; High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing
PubMed: 31619531
DOI: 10.1128/JCM.01315-19 -
Nature Biotechnology Sep 2021
Topics: Benchmarking; High-Throughput Nucleotide Sequencing; Reproducibility of Results; Software
PubMed: 34504352
DOI: 10.1038/s41587-021-01067-3 -
Archiwum Medycyny Sadowej I Kryminologii 2015The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on... (Review)
Review
The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on Sanger sequencing and continue to glowing these sciences based on Next generation sequencing (NGS). Next generation sequencing has excellent potential to flourish and increase the molecular applications in forensic sciences by jumping over the pitfalls of the conventional method of sequencing. The main advantages of NGS compared to conventional method that it utilizes simultaneously a large number of genetic markers with high-resolution of genetic data. These advantages will help in solving several challenges such as mixture analysis and dealing with minute degraded samples. Based on these new technologies, many markers could be examined to get important biological data such as age, geographical origins, tissue type determination, external visible traits and monozygotic twins identification. It also could get data related to microbes, insects, plants and soil which are of great medico-legal importance. Despite the dozens of forensic research involving NGS, there are requirements before using this technology routinely in forensic cases. Thus, there is a great need to more studies that address robustness of these techniques. Therefore, this work highlights the applications of forensic sciences in the era of massively parallel sequencing.
Topics: Forensic Genetics; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 27543959
DOI: 10.5114/amsik.2015.61029 -
Methods in Molecular Biology (Clifton,... 2023ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has gained wide popularity as a fast, straightforward, and efficient way of generating genome-wide...
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has gained wide popularity as a fast, straightforward, and efficient way of generating genome-wide maps of open chromatin and guiding identification of active regulatory elements and inference of DNA protein binding locations. Given the ubiquity of this method, uniform and standardized methods for processing and assessing the quality of ATAC-seq datasets are needed. Here, we describe the data processing pipeline used by the ENCODE (Encyclopedia of DNA Elements) consortium to process ATAC-seq data into peak call sets and signal tracks and to assess the quality of these datasets.
Topics: Chromatin Immunoprecipitation Sequencing; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Chromatin; DNA
PubMed: 36807076
DOI: 10.1007/978-1-0716-2899-7_17 -
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 -
Nature Biotechnology May 2024
Topics: Nanopore Sequencing; Humans; Nanopores; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 38760549
DOI: 10.1038/s41587-024-02231-1