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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 -
Nature Biotechnology May 2019Trajectory inference approaches analyze genome-wide omics data from thousands of single cells and computationally infer the order of these cells along developmental...
Trajectory inference approaches analyze genome-wide omics data from thousands of single cells and computationally infer the order of these cells along developmental trajectories. Although more than 70 trajectory inference tools have already been developed, it is challenging to compare their performance because the input they require and output models they produce vary substantially. Here, we benchmark 45 of these methods on 110 real and 229 synthetic datasets for cellular ordering, topology, scalability and usability. Our results highlight the complementarity of existing tools, and that the choice of method should depend mostly on the dataset dimensions and trajectory topology. Based on these results, we develop a set of guidelines to help users select the best method for their dataset. Our freely available data and evaluation pipeline ( https://benchmark.dynverse.org ) will aid in the development of improved tools designed to analyze increasingly large and complex single-cell datasets.
Topics: Benchmarking; Computational Biology; Genome; High-Throughput Nucleotide Sequencing; Single-Cell Analysis
PubMed: 30936559
DOI: 10.1038/s41587-019-0071-9 -
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 -
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 -
Briefings in Bioinformatics Nov 2013During the last 3 years, a number of approaches for the normalization of RNA sequencing data have emerged in the literature, differing both in the type of bias...
During the last 3 years, a number of approaches for the normalization of RNA sequencing data have emerged in the literature, differing both in the type of bias adjustment and in the statistical strategy adopted. However, as data continue to accumulate, there has been no clear consensus on the appropriate normalization method to be used or the impact of a chosen method on the downstream analysis. In this work, we focus on a comprehensive comparison of seven recently proposed normalization methods for the differential analysis of RNA-seq data, with an emphasis on the use of varied real and simulated datasets involving different species and experimental designs to represent data characteristics commonly observed in practice. Based on this comparison study, we propose practical recommendations on the appropriate normalization method to be used and its impact on the differential analysis of RNA-seq data.
Topics: High-Throughput Nucleotide Sequencing; Sequence Analysis, RNA
PubMed: 22988256
DOI: 10.1093/bib/bbs046 -
Nature Biotechnology Sep 2021
Topics: Benchmarking; High-Throughput Nucleotide Sequencing; Reproducibility of Results; Software
PubMed: 34504352
DOI: 10.1038/s41587-021-01067-3 -
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 -
Methods in Molecular Biology (Clifton,... 2015The pyrosequencing methodology was applied in 2005 by 454 Lifesciences to the emerging field of next generation sequencing (NGS), revolutionizing the way of DNA...
The pyrosequencing methodology was applied in 2005 by 454 Lifesciences to the emerging field of next generation sequencing (NGS), revolutionizing the way of DNA sequencing. In the last years the same strategy grew up and was technologically updated, reaching a high throughput in terms of amount of generated sequences (reads) per run and in terms of length of sequence up to values of 800-1,000 bases. These features of pyrosequencing perfectly fit to bacterial genome sequencing for the de novo assemblies and resequencing as well. The approaches of shotgun and paired ends sequencing allow the bacterial genome finishing providing a high-quality data in few days with unprecedented results.
Topics: Apyrase; Bacteria; Chromosome Mapping; DNA, Bacterial; DNA-Directed DNA Polymerase; Dideoxynucleotides; Genome, Bacterial; Genomic Library; High-Throughput Nucleotide Sequencing; Luciferases; Molecular Sequence Annotation; Polymerase Chain Reaction; Sequence Analysis, DNA; Sulfate Adenylyltransferase
PubMed: 25343858
DOI: 10.1007/978-1-4939-1720-4_4 -
Genes Jan 2019The adoption of single molecule real-time (SMRT) sequencing [...].
The adoption of single molecule real-time (SMRT) sequencing [...].
Topics: Animals; High-Throughput Nucleotide Sequencing; Humans; Plants; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 30621217
DOI: 10.3390/genes10010024 -
Genes Aug 2022Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of...
Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.
Topics: High-Throughput Nucleotide Sequencing; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction
PubMed: 36011328
DOI: 10.3390/genes13081418