-
Nature Biotechnology Dec 2023The first large genome fully sequenced by next-generation sequencing (NGS) was that of a bacteriophage using sequencing by synthesis (SBS) as a paradigm. SBS in NGS is... (Review)
Review
The first large genome fully sequenced by next-generation sequencing (NGS) was that of a bacteriophage using sequencing by synthesis (SBS) as a paradigm. SBS in NGS is underpinned by 'reversible-terminator chemistry'. To grow from proof of concept to being both affordable and practical, SBS needed to overcome a series of challenges, each of which required the invention of new chemistries. These included the design and synthesis of unnatural deoxynucleotide triphosphates (dNTPs), engineering a suitable polymerase, a new surface chemistry and an ingenious molecular solution to neutralize copying errors inherent to all polymerases. In this historical Perspective, we discuss how NGS was developed from Sanger sequencing, highlighting the chemistry behind this technology, which has impacted biology in unprecedented ways.
Topics: Sequence Analysis, DNA; Base Sequence; Genome; High-Throughput Nucleotide Sequencing
PubMed: 37845570
DOI: 10.1038/s41587-023-01986-3 -
Nature Biotechnology Nov 2023Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop...
Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.
Topics: Humans; Animals; Mice; Microfluidics; High-Throughput Nucleotide Sequencing; Single-Cell Analysis; Genomics; Transcriptome
PubMed: 36879006
DOI: 10.1038/s41587-023-01685-z -
Molecular Microbiology Aug 2023Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid... (Review)
Review
Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high-throughput functional genomics, focusing primarily on transposon (Tn)-based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.
Topics: Humans; DNA Transposable Elements; Genomics; Mutagenesis; Mycobacterium tuberculosis; Phenotype; Genome, Bacterial; High-Throughput Nucleotide Sequencing
PubMed: 37278255
DOI: 10.1111/mmi.15103 -
Applied and Environmental Microbiology Oct 2023The long-read amplicon provides a species-level solution for the community. With the improvement of nanopore flowcells, the accuracy of Oxford Nanopore Technologies...
The long-read amplicon provides a species-level solution for the community. With the improvement of nanopore flowcells, the accuracy of Oxford Nanopore Technologies (ONT) R10.4.1 has been substantially enhanced, with an average of approximately 99%. To evaluate its effectiveness on amplicons, three types of microbiomes were analyzed by 16S ribosomal RNA (hereinafter referred to as "16S") amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1) in the current study. We showed the error rate, recall, precision, and bias index in the mock sample. The error rate of ONT R10.4.1 was greatly reduced, with a better recall in the case of the synthetic community. Meanwhile, in different types of environmental samples, ONT R10.4.1 analysis resulted in a composition similar to Pacbio data. We found that classification tools and databases influence ONT data. Based on these results, we conclude that the ONT R10.4.1 16S amplicon can also be used for application in environmental samples. IMPORTANCE The long-read amplicon supplies the community with a species-level solution. Due to the high error rate of nanopore sequencing early on, it has not been frequently used in 16S studies. Oxford Nanopore Technologies (ONT) introduced the R10.4.1 flowcell with Q20+ reagent to achieve more than 99% accuracy as sequencing technology advanced. However, there has been no published study on the performance of commercial PromethION sequencers with R10.4.1 flowcells on 16S sequencing or on the impact of accuracy improvement on taxonomy (R9.4.1 to R10.4.1) using 16S ONT data. In this study, three types of microbiomes were investigated by 16S ribosomal RNA (rRNA) amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1). In the mock sample, we displayed the error rate, recall, precision, and bias index. We observed that the error rate in ONT R10.4.1 is significantly lower, especially when deletions are involved. First and foremost, R10.4.1 and Pacific Bioscience platforms reveal a similar microbiome in environmental samples. This study shows that the R10.4.1 full-length 16S rRNA sequences allow for species identification of environmental microbiota.
Topics: RNA, Ribosomal, 16S; Sequence Analysis, DNA; Nanopores; Microbiota; High-Throughput Nucleotide Sequencing
PubMed: 37800969
DOI: 10.1128/aem.00605-23 -
Gene Apr 2024Neuronal development is a highly regulated mechanism that is central to organismal function in animals. In humans, disruptions to this process can lead to a range of... (Review)
Review
Neuronal development is a highly regulated mechanism that is central to organismal function in animals. In humans, disruptions to this process can lead to a range of neurodevelopmental phenotypes, including Schizophrenia (SCZ). SCZ has a significant genetic component, whereby an individual with an SCZ affected family member is eight times more likely to develop the disease than someone with no family history of SCZ. By examining a combination of genomic, transcriptomic and epigenomic datasets, large-scale 'omics' studies aim to delineate the relationship between genetic variation and abnormal cellular activity in the SCZ brain. Herein, we provide a brief overview of some of the key omics methods currently being used in SCZ research, including RNA-seq, the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and high-throughput chromosome conformation capture (3C) approaches (e.g., Hi-C), as well as single-cell/nuclei iterations of these methods. We also discuss how these techniques are being employed to further our understanding of the genetic basis of SCZ, and to identify associated molecular pathways, biomarkers, and candidate drug targets.
Topics: Animals; Humans; Schizophrenia; Chromatin; Chromatin Immunoprecipitation Sequencing; Brain; High-Throughput Nucleotide Sequencing
PubMed: 38266791
DOI: 10.1016/j.gene.2024.148198 -
Trends in Genetics : TIG Jan 2024Recent technological and algorithmic advances enable single-cell transcriptomic analysis with remarkable depth and breadth. Nonetheless, a persistent challenge is the... (Review)
Review
Recent technological and algorithmic advances enable single-cell transcriptomic analysis with remarkable depth and breadth. Nonetheless, a persistent challenge is the compromise between the ability to profile high numbers of cells and the achievement of full-length transcript coverage. Currently, the field is progressing and developing new and creative solutions that improve cellular throughput, gene detection sensitivity and full-length transcript capture. Furthermore, long-read sequencing approaches for single-cell transcripts are breaking frontiers that have previously blocked full transcriptome characterization. We here present a comprehensive overview of available options for single-cell transcriptome profiling, highlighting the key advantages and disadvantages of each approach.
Topics: Transcriptome; High-Throughput Nucleotide Sequencing; Gene Expression Profiling; Sequence Analysis, RNA
PubMed: 37953195
DOI: 10.1016/j.tig.2023.10.003 -
Human Genomics Aug 2023Long-read DNA sequencing technologies have been rapidly evolving in recent years, and their ability to assess large and complex regions of the genome makes them ideal... (Review)
Review
Long-read DNA sequencing technologies have been rapidly evolving in recent years, and their ability to assess large and complex regions of the genome makes them ideal for clinical applications in molecular diagnosis and therapy selection, thereby providing a valuable tool for precision medicine. In the third-generation sequencing duopoly, Oxford Nanopore Technologies and Pacific Biosciences work towards increasing the accuracy, throughput, and portability of long-read sequencing methods while trying to keep costs low. These trades have made long-read sequencing an attractive tool for use in research and clinical settings. This article provides an overview of current clinical applications and limitations of long-read sequencing and explores its potential for point-of-care testing and health care in remote settings.
Topics: Humans; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Nanopores
PubMed: 37553611
DOI: 10.1186/s40246-023-00522-3 -
Cancer Treatment Reviews Sep 2023Cancer has become a global health issue and liquid biopsy has emerged as a non-invasive tool for various applications. In cancer, circulating tumor DNA (ctDNA) can be... (Review)
Review
Cancer has become a global health issue and liquid biopsy has emerged as a non-invasive tool for various applications. In cancer, circulating tumor DNA (ctDNA) can be detected from cell-free DNA (cfDNA) obtained from plasma and has potential for early diagnosis, treatment, resistance, minimal residual disease detection, and tumoral heterogeneity identification. However, the low frequency of ctDNA requires techniques for accurate analysis. Multitarget assay such as Next Generation Sequencing (NGS) need improvement to achieve limits of detection that can identify the low frequency variants present in the cfDNA. In this review, we provide a general overview of the use of cfDNA and ctDNA in cancer, and discuss techniques developed to optimize NGS as a tool for ctDNA detection. We also summarize the results obtained using NGS strategies in both investigational and clinical contexts.
Topics: Humans; Circulating Tumor DNA; High-Throughput Nucleotide Sequencing; Mutation; Cell-Free Nucleic Acids; Neoplasms; Biomarkers, Tumor
PubMed: 37390697
DOI: 10.1016/j.ctrv.2023.102595 -
Nature Reviews. Genetics Sep 2023The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read... (Review)
Review
The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein-DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.
Topics: Humans; Sequence Analysis, DNA; DNA Copy Number Variations; High-Throughput Nucleotide Sequencing; Genome, Human; Technology
PubMed: 37161088
DOI: 10.1038/s41576-023-00600-1 -
Trends in Biotechnology Dec 2023The impact of next-generation sequencing (NGS) cannot be overestimated. The technology has transformed the field of life science, contributing to a dramatic expansion in... (Review)
Review
The impact of next-generation sequencing (NGS) cannot be overestimated. The technology has transformed the field of life science, contributing to a dramatic expansion in our understanding of human health and disease and our understanding of biology and ecology. The vast majority of the major NGS systems today are based on the concept of 'sequencing by synthesis' (SBS) with sequential detection of nucleotide incorporation using an engineered DNA polymerase. Based on this strategy, various alternative platforms have been developed, including the use of either native nucleotides or reversible terminators and different strategies for the attachment of DNA to a solid support. In this review, some of the key concepts leading to this remarkable development are discussed.
Topics: Humans; Sequence Analysis, DNA; DNA; Nucleotides; DNA-Directed DNA Polymerase; High-Throughput Nucleotide Sequencing
PubMed: 37482467
DOI: 10.1016/j.tibtech.2023.06.007