-
Methods in Molecular Biology (Clifton,... 2017Epigenetic modifications, such as DNA methylation, can contribute to gene regulation and chromosomal stability. There are several methods and techniques available for...
Epigenetic modifications, such as DNA methylation, can contribute to gene regulation and chromosomal stability. There are several methods and techniques available for methylation analysis, ranging from global methylation to gene-specific targeted regions. Bisulfite conversion enables numerous methodologies to be used for downstream applications, including pyrosequencing which measures DNA methylation at an individual CpG site level. This allows specific regions of interest to be targeted for DNA methylation analysis. Designing and optimizing pyrosequencing assays correctly is vital for the interpretation of results.Dysregulation of DNA methylation has been implicated in human diseases, with regions such as repeat elements commonly altered. Human population studies investigating these tend to use consensus sequences to target repeat elements. However, these elements have high mutational rates, particularly Alu sequences, which could lead to assay bias and masking of changes at a regional level. Therefore, it may be more beneficial to target specific repeat elements depending upon their chromosomal location, rather than analyzing overall methylation levels.
Topics: DNA Methylation; Epigenomics; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Sulfites
PubMed: 26246355
DOI: 10.1007/7651_2015_285 -
Expert Review of Molecular Diagnostics Sep 2016The review highlights the impact of next-generation sequencing (NGS) on genomic medicine and the consequences of the progression from a single-gene panel technology to a... (Review)
Review
INTRODUCTION
The review highlights the impact of next-generation sequencing (NGS) on genomic medicine and the consequences of the progression from a single-gene panel technology to a whole exome sequencing approach.
AREAS COVERED
We brought together literature-based evidences, personal unpublished data and clinical experience to provide a critical overview of the impact of NGS on our daily clinical practice. Expert commentary: NGS has changed the role of clinical geneticist and has broadened the view accomplishing a transition from a monogenic Mendelian perspective to an oligogenic approach to disorders. Thus, it is a compelling new expertise which combines clinical evaluation with big omics data interpretation and moves forward to phenotype re-evaluation in light of data analysis. We introduced the term, 'exotyping', to highlight this holistic approach. Further, the review discusses the impact that the combination of genetic reprogramming and transcriptome analysis will have on the discovery of evidence-based therapies.
Topics: Genomics; High-Throughput Nucleotide Sequencing; Humans; Molecular Medicine
PubMed: 27574853
DOI: 10.1080/14737159.2016.1224181 -
The Journal of Molecular Diagnostics :... Feb 2020Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are...
Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.
Topics: Computational Biology; DNA Contamination; Databases, Nucleic Acid; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Reference Standards; Reproducibility of Results; Sequence Analysis, DNA
PubMed: 31837431
DOI: 10.1016/j.jmoldx.2019.10.011 -
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 -
Methods in Molecular Biology (Clifton,... 2018Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a...
Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a given tissue and sample type. NGS analysis requires high-quality RNA extraction and preparation of microRNA libraries. In this chapter, we describe the methods used for RNA extraction from tissue specimens, serum, cytological slides, and formalin-fixed paraffin-embedded samples. Although the described library preparation and sequencing approaches are based on Illumina NextSeq 500 sequencing technology, the presented principles shall be compatible with other commercially available sequencing platforms.
Topics: Animals; Gene Library; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs
PubMed: 29959676
DOI: 10.1007/978-1-4939-8624-8_8 -
Molecular Biology and Evolution Aug 2019Advances in sequencing technology have resulted in the expectation that genomic studies will become more representative of organismal diversity. To test this...
Advances in sequencing technology have resulted in the expectation that genomic studies will become more representative of organismal diversity. To test this expectation, we explored species representation of nonhuman eukaryotes in the Sequence Read Archive. Though species richness has been increasing steadily, species evenness is decreasing over time. Moreover, the top 1% most studied organisms increasingly represent a larger proportion of total experiments, demonstrating growing bias in favor of a small minority of species. To better understand molecular processes and patterns, genomic studies should reverse current trends by adopting more comparative approaches.
Topics: Animals; Genomics; High-Throughput Nucleotide Sequencing
PubMed: 31077317
DOI: 10.1093/molbev/msz117 -
Trends in Molecular Medicine Jun 2017Single-cell RNA sequencing (scRNA-seq) is an exciting new technology allowing the analysis of transcriptomes from individual cells, and is ideally suited to address the... (Review)
Review
Single-cell RNA sequencing (scRNA-seq) is an exciting new technology allowing the analysis of transcriptomes from individual cells, and is ideally suited to address the inherent complexity and dynamics of the central nervous system. scRNA-seq has already been applied to the study of molecular taxonomy of the brain. These works have paved the way to expanding our understanding of the nervous system and provide insights into cellular susceptibilities and molecular mechanisms in neurological and neurodegenerative diseases. We discuss recent progress and challenges in applying this technology to advance our understanding of the brain. We advocate the application of scRNA-seq in the discovery of targets and biomarkers as a new approach in developing novel therapeutics for the treatment of neurodegenerative diseases.
Topics: Animals; Biomarkers; High-Throughput Nucleotide Sequencing; Humans; Neurodegenerative Diseases; Sequence Analysis, RNA
PubMed: 28501348
DOI: 10.1016/j.molmed.2017.04.006 -
British Journal of Haematology Mar 2013Next-generation sequencing platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumour... (Review)
Review
Next-generation sequencing platforms have evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumour specimens. Here, we review the feasibility and practicality of this novel laboratory technology. In particular, we focus on the utility of next-generation sequencing technology in characterizing haematological neoplasms and the landmark findings in key haematological malignancies. We also discuss deep-sequencing strategies to analyse the constantly increasing number of molecular markers applied for disease classification, patient stratification and individualized monitoring of minimal residual disease. Although many facets of this assay need to be taken into account, amplicon deep-sequencing has already demonstrated a promising technical performance and is being continuously developed towards routine application in diagnostic laboratories so that an impact on clinical practice can be achieved.
Topics: Hematology; High-Throughput Nucleotide Sequencing; Humans; Molecular Diagnostic Techniques; Sequence Analysis, DNA
PubMed: 23294427
DOI: 10.1111/bjh.12194 -
Expert Review of Molecular Diagnostics 2016The huge parallel sequencing capabilities of next generation sequencing technologies have made them the tools of choice to characterize genomic aberrations for research... (Review)
Review
The huge parallel sequencing capabilities of next generation sequencing technologies have made them the tools of choice to characterize genomic aberrations for research and diagnostic purposes. For clinical applications, screening the whole genome or exome is challenging owing to the large genomic area to be sequenced, associated costs, complexity of data, and lack of known clinical significance of all genes. Consequently, routine screening involves limited markers with established clinical relevance. This process, referred to as targeted genome sequencing, requires selective enrichment of the genomic areas comprising these markers via one of several primer or probe-based enrichment strategies, followed by sequencing of the enriched genomic areas. Here, the authors review current target enrichment approaches and next generation sequencing platforms, focusing on the underlying principles, capabilities, and limitations of each technology along with validation and implementation for clinical testing.
Topics: High-Throughput Nucleotide Sequencing; Molecular Diagnostic Techniques; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 26680590
DOI: 10.1586/14737159.2016.1133298 -
Nature Feb 2023
Topics: High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Inventions
PubMed: 36807670
DOI: 10.1038/d41586-023-00512-4