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Molecular Aspects of Medicine Apr 2024Current precision cancer medicine is dependent on the analyses of a plethora of clinically relevant genomic aberrations. During the last decade, next-generation... (Review)
Review
Current precision cancer medicine is dependent on the analyses of a plethora of clinically relevant genomic aberrations. During the last decade, next-generation sequencing (NGS) has gradually replaced most other methods for precision cancer diagnostics, spanning from targeted tumor-informed assays and gene panel sequencing to global whole-genome and whole-transcriptome sequencing analyses. The shift has been impelled by a clinical need to assess an increasing number of genomic alterations with diagnostic, prognostic and predictive impact, including more complex biomarkers (e.g. microsatellite instability, MSI, and homologous recombination deficiency, HRD), driven by the parallel development of novel targeted therapies and enabled by the rapid reduction in sequencing costs. This review focuses on these sequencing-based methods, puts their emergence in a historic perspective, highlights their clinical utility in diagnostics and decision-making in pediatric and adult cancer, as well as raises challenges for their clinical implementation. Finally, the importance of applying sensitive tools for longitudinal monitoring of treatment response and detection of measurable residual disease, as well as future avenues in the rapidly evolving field of sequencing-based methods are discussed.
Topics: Humans; Child; Mutation; Neoplasms; Genomics; High-Throughput Nucleotide Sequencing; Precision Medicine
PubMed: 38330674
DOI: 10.1016/j.mam.2024.101250 -
Journal of Translational Medicine Jan 2024The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics.... (Review)
Review
The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics. However, a new era has emerged with the advent of long-read sequencing (LRS), which offers substantial improvements over its predecessor, short-read sequencing (SRS). LRS produces reads that are several kilobases long, enabling researchers to obtain more complete and contiguous genomic information, characterize structural variations, and study epigenetic modifications. The current leaders in LRS technologies are Pacific Biotechnologies (PacBio) and Oxford Nanopore Technologies (ONT), each offering a distinct set of advantages. This review covers the workflow of long-read metagenomics sequencing, including sample preparation (sample collection, sample extraction, and library preparation), sequencing, processing (quality control, assembly, and binning), and analysis (taxonomic annotation and functional annotation). Each section provides a concise outline of the key concept of the methodology, presenting the original concept as well as how it is challenged or modified in the context of LRS. Additionally, the section introduces a range of tools that are compatible with LRS and can be utilized to execute the LRS process. This review aims to present the workflow of metagenomics, highlight the transformative impact of LRS, and provide researchers with a selection of tools suitable for this task.
Topics: RNA, Ribosomal, 16S; High-Throughput Nucleotide Sequencing; Metagenomics; Sequence Analysis, DNA; Genomics
PubMed: 38282030
DOI: 10.1186/s12967-024-04917-1 -
Methods in Molecular Biology (Clifton,... 2024Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including...
Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including single-nucleotide polymorphisms (SNPs). These SNPs can be utilized to gain evolutionary insights into patterns observed in DNA barcodes, to infer population structure and phylogenies, to detect gene flow and introgression, and to perform species delimitation analyses. The protocol includes chemically shearing genomic DNA with restriction enzymes, unique tagging, size selection, and amplification of the resulting DNA fragments. Here we provide a detailed description of each step of the protocol, as well as information on essential equipment and common issues encountered during laboratory work.
Topics: DNA Barcoding, Taxonomic; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; DNA; Gene Library; Humans
PubMed: 38683321
DOI: 10.1007/978-1-0716-3581-0_13 -
Nature Communications Jan 2024The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a...
The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compare the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis reveals substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also consider the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produce the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results lead to widespread improvements across AoU.
Topics: Humans; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Genome, Human; Population Health; INDEL Mutation
PubMed: 38281971
DOI: 10.1038/s41467-024-44804-3 -
Molecular Aspects of Medicine Apr 2024Single-cell technologies have transformed biomedical research over the last decade, opening up new possibilities for understanding cellular heterogeneity, both at the... (Review)
Review
Single-cell technologies have transformed biomedical research over the last decade, opening up new possibilities for understanding cellular heterogeneity, both at the genomic and transcriptomic level. In addition, more recent developments of spatial transcriptomics technologies have made it possible to profile cells in their tissue context. In parallel, there have been substantial advances in sequencing technologies, and the third generation of methods are able to produce reads that are tens of kilobases long, with error rates matching the second generation short reads. Long reads technologies make it possible to better map large genome rearrangements and quantify isoform specific abundances. This further improves our ability to characterize functionally relevant heterogeneity. Here, we show how researchers have begun to combine single-cell, spatial transcriptomics, and long-read technologies, and how this is resulting in powerful new approaches to profiling both the genome and the transcriptome. We discuss the achievements so far, and we highlight remaining challenges and opportunities.
Topics: Humans; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Genomics; Gene Expression Profiling; Transcriptome
PubMed: 38368637
DOI: 10.1016/j.mam.2024.101255 -
Medical Principles and Practice :... 2024The success in determining the whole genome sequence of a bacterial pathogen was first achieved in 1995 by determining the complete nucleotide sequence of Haemophilus... (Review)
Review
The success in determining the whole genome sequence of a bacterial pathogen was first achieved in 1995 by determining the complete nucleotide sequence of Haemophilus influenzae Rd using the chain-termination method established by Sanger et al. in 1977 and automated by Hood et al. in 1987. However, this technology was laborious, costly, and time-consuming. Since 2004, high-throughput next-generation sequencing technologies have been developed, which are highly efficient, require less time, and are cost-effective for whole genome sequencing (WGS) of all organisms, including bacterial pathogens. In recent years, the data obtained using WGS technologies coupled with bioinformatics analyses of the sequenced genomes have been projected to revolutionize clinical bacteriology. WGS technologies have been used in the identification of bacterial species, strains, and genotypes from cultured organisms and directly from clinical specimens. WGS has also helped in determining resistance to antibiotics by the detection of antimicrobial resistance genes and point mutations. Furthermore, WGS data have helped in the epidemiological tracking and surveillance of pathogenic bacteria in healthcare settings as well as in communities. This review focuses on the applications of WGS in clinical bacteriology.
Topics: Humans; Whole Genome Sequencing; Genome, Bacterial; Drug Resistance, Bacterial; High-Throughput Nucleotide Sequencing
PubMed: 38402870
DOI: 10.1159/000538002 -
Journal of Computational Biology : a... Dec 2023Processing large data sets has become an essential part of computational genomics. Greatly increased availability of sequence data from multiple sources has fueled... (Review)
Review
Processing large data sets has become an essential part of computational genomics. Greatly increased availability of sequence data from multiple sources has fueled breakthroughs in genomics and related fields but has led to computational challenges processing large sequencing experiments. The minimizer sketch is a popular method for sequence sketching that underlies core steps in computational genomics such as read mapping, sequence assembling, k-mer counting, and more. In most applications, minimizer sketches are constructed using one of few classical approaches. More recently, efforts have been put into building minimizer sketches with desirable properties compared with the classical constructions. In this survey, we review the history of the minimizer sketch, the theories developed around the concept, and the plethora of applications taking advantage of such sketches. We aim to provide the readers a comprehensive picture of the research landscape involving minimizer sketches, in anticipation of better fusion of theory and application in the future.
Topics: Algorithms; Sequence Analysis, DNA; Genomics; High-Throughput Nucleotide Sequencing; Software
PubMed: 37646787
DOI: 10.1089/cmb.2023.0094 -
Critical Reviews in Microbiology Nov 2023High-throughput DNA sequencing-based approaches continue to revolutionise our understanding of microbial ecosystems, including those associated with fermented foods.... (Review)
Review
High-throughput DNA sequencing-based approaches continue to revolutionise our understanding of microbial ecosystems, including those associated with fermented foods. Metagenomic and metatranscriptomic approaches are state-of-the-art biological profiling methods and are employed to investigate a wide variety of characteristics of microbial communities, such as taxonomic membership, gene content and the range and level at which these genes are expressed. Individual groups and consortia of researchers are utilising these approaches to produce increasingly large and complex datasets, representing vast populations of microorganisms. There is a corresponding requirement for the development and application of appropriate bioinformatic tools and pipelines to interpret this data. This review critically analyses the tools and pipelines that have been used or that could be applied to the analysis of metagenomic and metatranscriptomic data from fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied, to highlight the insights that these approaches can provide.
Topics: Microbiota; Metagenome; Computational Biology; Fermented Foods; High-Throughput Nucleotide Sequencing
PubMed: 36287644
DOI: 10.1080/1040841X.2022.2132850 -
Clinical Chemistry Sep 2023Oxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare...
BACKGROUND
Oxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare long-template libraries for long-read TGS, particularly the ONT method for analysis of hemoglobinopathy variants involving complex structures and occurring in GC-rich and/or homologous regions.
METHODS
A multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons for HBA2/1, HBG2/1, HBD, and HBB, as well as the allelic amplicons for targeted deletions and special structural variations. Library construction was performed using long-PCR products, and sequencing was conducted on an Oxford Nanopore MinION instrument. Genotypes were identified based on integrative genomics viewer (IGV) plots.
RESULTS
This novel long-read TGS method distinguished all single nucleotide variants and structural variants within HBA2/1, HBG2/1, HBD, and HBB based on the whole-gene sequence reads. Targeted deletions and special structural variations were also identified according to the specific allelic reads. The result of 158 α-/β-thalassemia samples showed 100% concordance with previously known genotypes.
CONCLUSIONS
This ONT TGS method is high-throughput, which can be used for molecular screening and genetic diagnosis of hemoglobinopathies. The strategy of multiplex long PCR is an efficient strategy for library preparation, providing a practical reference for TGS assay development.
Topics: Humans; Sequence Analysis, DNA; Nanopores; Genomics; Hemoglobinopathies; Gene Library; High-Throughput Nucleotide Sequencing
PubMed: 37311260
DOI: 10.1093/clinchem/hvad073 -
Journal of Vascular and Interventional... Aug 2023The discovery of increasing numbers of actionable molecular and gene targets for cancer treatment has driven the demand for tissue sampling for next-generation... (Review)
Review
The discovery of increasing numbers of actionable molecular and gene targets for cancer treatment has driven the demand for tissue sampling for next-generation sequencing (NGS). Requirements for sequencing can be very specific, and inadequate sampling leads to delays in management and decision making. It is important that interventional radiologists are aware of NGS technologies and their common applications and be cognizant of the factors that contribute to successful sample sequencing. This review summarizes the fundamentals of cancer tissue collection and processing for NGS. It elaborates on sequencing technologies and their applications with the aim of providing readers with a working understanding that can enhance their clinical practice. It then describes imaging, tumor, biopsy, and sample collection factors that improve the chances of NGS success. Finally, it discusses future practice, highlighting the problem of undersampling in both clinical and research settings and the opportunities within interventional radiology to address this.
Topics: Humans; Neoplasms; Biopsy; High-Throughput Nucleotide Sequencing
PubMed: 36977432
DOI: 10.1016/j.jvir.2023.03.012