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Revue Des Maladies Respiratoires Apr 2023Genetic diagnoses have progressed through the development of Next Generation Sequencing (NGS), which enables improved patient care and more precise genetic counseling.... (Review)
Review
Genetic diagnoses have progressed through the development of Next Generation Sequencing (NGS), which enables improved patient care and more precise genetic counseling. NGS techniques analyze DNA regions of interest in view accurately determining the relevant nucleotide sequence. Different kinds of analysis apply NGS : multigene panel testing, Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS). While regions of interest depend on the type of analysis (multigene panels testing studies the exons of genes implicated in a particular phenotype, WES studies all exons of all genes, and WGS studies all exons and introns), the technical protocol remains similar. Clinical/biological interpretation is based on a body of evidence allowing categorization of variants into five groups (from benign to pathogenic) in accordance with an international classification, which takes into account segregation criteria (variant detected in affected relatives, but absent in healthy relatives), matching phenotype, databases, scientific literature, prediction scores and data drawn from functional studies. Clinical/biological interaction and expertise are essential during this interpretative step. Pathogenic and probably pathogenic variants are returned to the clinician. Variants of unknown significance can likewise be returned, if they are liable to be reclassified through further analysis as pathogenic or benign. Variant classifications may change, as new data emerge suggesting or ruling out pathogenicity.
Topics: Humans; High-Throughput Nucleotide Sequencing; Phenotype; Exons
PubMed: 36863993
DOI: 10.1016/j.rmr.2023.01.026 -
Methods in Molecular Biology (Clifton,... 2021The NovaSeq 6000 is a sequencing platform from Illumina that enables the sequencing of short reads with an output up to 6 Tb. The NovaSeq 6000 uses the typical Illumina...
The NovaSeq 6000 is a sequencing platform from Illumina that enables the sequencing of short reads with an output up to 6 Tb. The NovaSeq 6000 uses the typical Illumina sequencing workflow based on library preparation, cluster generation by in situ amplification, and sequencing by synthesis. Flexibility is one of the major features of the NovaSeq 6000. Several types of sequencing kits coupled with dual flow cell mode enable high scalability of sequencing outputs to match a wide range of applications from complete genome sequencing to metagenomics analysis. In this chapter, after explaining how to assemble a normalized pool of libraries for sequencing, we will describe the experimental steps required to run the pools on the NovaSeq 6000 platform.
Topics: Genomic Library; Genomics; High-Throughput Nucleotide Sequencing; Research Design; Sequence Analysis, DNA; Workflow
PubMed: 33961215
DOI: 10.1007/978-1-0716-1099-2_2 -
Nature Methods Feb 2021High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing...
High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus sequencing, yielding high-accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence ribosomal RNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities in which we observed a chimera rate <0.02%. To reach a mean UMI consensus error rate <0.01%, a UMI read coverage of 15× (ONT R10.3), 25× (ONT R9.4.1) and 3× (Pacific Biosciences circular consensus sequencing) is needed, which provides a mean error rate of 0.0042%, 0.0041% and 0.0007%, respectively.
Topics: High-Throughput Nucleotide Sequencing; Microbiota; Nanopores; Workflow
PubMed: 33432244
DOI: 10.1038/s41592-020-01041-y -
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 -
Nature Methods Jun 2021Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable...
Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed 'single-cell combinatorial fluidic indexing' (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation.
Topics: Animals; Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats; Cost-Benefit Analysis; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Humans; Mice; Microfluidics; Receptors, Antigen, T-Cell; Single-Cell Analysis; Transcriptome
PubMed: 34059827
DOI: 10.1038/s41592-021-01153-z -
STAR Protocols Sep 2021The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of...
The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs. Custom biotin RNAs are added and biotinylated RNAs are isolated for generation of cDNA libraries. The sequencing data are normalized to controls for quantitative assessment of the nascent transcriptome. The protocol takes 4 days, not including sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Palozola et al. (2017).
Topics: Biotin; Cell Line; Chemical Precipitation; High-Throughput Nucleotide Sequencing; Humans; Molecular Biology; RNA; RNA-Seq; Uridine
PubMed: 34485932
DOI: 10.1016/j.xpro.2021.100651 -
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 -
Clinical Laboratory Aug 2023Next-generation sequencing (NGS) methods have become more commonly performed in clinical and research laboratories. (Review)
Review
BACKGROUND
Next-generation sequencing (NGS) methods have become more commonly performed in clinical and research laboratories.
METHODS
This review summarizes the current laboratory NGS-based diagnostic approaches in pharmacogenomics including targeted multi-gene panel sequencing, whole-exome sequencing (WES), and whole-genome sequencing (WGS).
RESULTS
Clinical laboratories perform multiple non-uniform types of pharmacogenetic panels, which can reduce the overall number of single-gene tests to be more cost-efficient. Compared to the targeted multi-gene panels, which are not typically designed to detect novel variants, WES and WGS have a greater potential to identify secondary pharmacogenomic findings, which might be predictive for the pharmacotherapy outcome of different patient settings. WGS overcomes the limitations of WES enabling a more accurate exome-sequencing at appropriate coverage and the sequencing of non-coding regions. Different NGS-based study designs with different test strategies and study populations, varying sample sizes, and distinct analytical and interpretation procedures lead to different identification results of pharmacogenomic variants.
CONCLUSIONS
The rapid progress in gene sequencing technologies will overcome the clinical and laboratory challenges of WES and WGS. Further high throughput NGS-based pharmacogenomics studies in different populations and patient settings are necessary to expand knowledge about rare functional variants and to enhance translation in clinical practice.
Topics: Humans; Pharmacogenetics; High-Throughput Nucleotide Sequencing
PubMed: 37560847
DOI: 10.7754/Clin.Lab.2023.230103 -
Digestive Diseases and Sciences Mar 2020Over the past decade, it has become exceedingly clear that the microbiome is a critical factor in human health and disease and thus should be investigated to develop... (Review)
Review
Over the past decade, it has become exceedingly clear that the microbiome is a critical factor in human health and disease and thus should be investigated to develop innovative treatment strategies. The field of metagenomics has come a long way in leveraging the advances of next-generation sequencing technologies resulting in the capability to identify and quantify all microorganisms present in human specimens. However, the field of metagenomics is still in its infancy, specifically in regard to the limitations in computational analysis, statistical assessments, standardization, and validation due to vast variability in the cohorts themselves, experimental design, and bioinformatic workflows. This review summarizes the methods, technologies, computational tools, and model systems for characterizing and studying the microbiome. We also discuss important considerations investigators must make when interrogating the involvement of the microbiome in health and disease in order to establish robust results and mechanistic insights before moving into therapeutic design and intervention.
Topics: Animals; Computational Biology; High-Throughput Nucleotide Sequencing; Humans; Machine Learning; Metagenomics; Microbiota; Sequence Analysis, DNA
PubMed: 32002757
DOI: 10.1007/s10620-020-06091-y