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Nature Microbiology May 2023Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states...
Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.
Topics: Transcriptome; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing
PubMed: 37012420
DOI: 10.1038/s41564-023-01348-4 -
Genome Research May 2023Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and...
Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.
Topics: Genome, Bacterial; Sequence Analysis, DNA; Plasmids; High-Throughput Nucleotide Sequencing; Proteomics
PubMed: 37156622
DOI: 10.1101/gr.277369.122 -
Science Progress Dec 2019Conventional microbiological water monitoring uses culture-dependent techniques to screen indicator microbial species such as and fecal coliforms. With high-throughput,... (Review)
Review
Conventional microbiological water monitoring uses culture-dependent techniques to screen indicator microbial species such as and fecal coliforms. With high-throughput, second-generation sequencing technologies becoming less expensive, water quality monitoring programs can now leverage the massively parallel nature of second-generation sequencing technologies for batch sample processing to simultaneously obtain compositional and functional information of culturable and as yet uncultured microbial organisms. This review provides an introduction to the technical capabilities and considerations necessary for the use of second-generation sequencing technologies, specifically 16S rDNA amplicon and whole-metagenome sequencing, to investigate the composition and functional potential of microbiomes found in water and wastewater systems.
Topics: Bacteria; DNA, Bacterial; High-Throughput Nucleotide Sequencing; Wastewater; Water Microbiology
PubMed: 31818206
DOI: 10.1177/0036850419881855 -
Scientific Reports Oct 2023Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random...
Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.
Topics: High-Throughput Nucleotide Sequencing; Antibodies; Epitopes
PubMed: 37884618
DOI: 10.1038/s41598-023-45538-w -
Frontiers in Cellular and Infection... 2022We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from...
We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from 51 patients. We compared targeted NGS to Sanger sequencing in clinical samples submitted for Sanger sequencing. The overall concordance rate was 58% (32/55) for NGS vs. Sanger. NGS identified 9 polymicrobial and 2 monomicrobial infections among 19 Sanger-negative samples and 8 polymicrobial infections in 11 samples where a 16S gene was identified by gel electrophoresis, but could not be mapped to an identified pathogen by Sanger. We estimated that NGS could have contributed to patient management in 6/18 evaluated patients and thus has an advantage over Sanger sequencing in certain polymicrobial infections.
Topics: Humans; High-Throughput Nucleotide Sequencing; Coinfection; Mutation
PubMed: 36339334
DOI: 10.3389/fcimb.2022.955481 -
Trends in Genetics : TIG Mar 2022Nanopore sequencing provides signal data corresponding to the nucleotide motifs sequenced. Through machine learning-based methods, these signals are translated into... (Review)
Review
Nanopore sequencing provides signal data corresponding to the nucleotide motifs sequenced. Through machine learning-based methods, these signals are translated into long-read sequences that overcome the read size limit of short-read sequencing. However, analyzing the raw nanopore signal data provides many more opportunities beyond just sequencing genomes and transcriptomes: algorithms that use machine learning approaches to extract biological information from these signals allow the detection of DNA and RNA modifications, the estimation of poly(A) tail length, and the prediction of RNA secondary structures. In this review, we discuss how developments in machine learning methodologies contributed to more accurate basecalling and lower error rates, and how these methods enable new biological discoveries. We argue that direct nanopore sequencing of DNA and RNA provides a new dimensionality for genomics experiments and highlight challenges and future directions for computational approaches to extract the additional information provided by nanopore signal data.
Topics: Algorithms; Genomics; High-Throughput Nucleotide Sequencing; Machine Learning; Nanopore Sequencing; Nanopores; Sequence Analysis, DNA
PubMed: 34711425
DOI: 10.1016/j.tig.2021.09.001 -
Anesthesia and Analgesia Aug 2020Perioperative medicine is changing from a "protocol-based" approach to a progressively personalized care model. New molecular techniques and comprehensive perioperative... (Review)
Review
Perioperative medicine is changing from a "protocol-based" approach to a progressively personalized care model. New molecular techniques and comprehensive perioperative medical records allow for detection of patient-specific phenotypes that may better explain, or even predict, a patient's response to perioperative stress and anesthetic care. Basic science technology has significantly evolved in recent years with the advent of powerful approaches that have translational relevance. It is incumbent on us as a primarily clinical specialty to have an in-depth understanding of rapidly evolving underlying basic science techniques to incorporate such approaches into our own research, critically interpret the literature, and improve future anesthesia patient care. This review focuses on 3 important and most likely practice-changing basic science techniques: next-generation sequencing (NGS), clustered regularly interspaced short palindromic repeat (CRISPR) modulations, and inducible pluripotent stem cells (iPSCs). Each technique will be described, potential advantages and limitations discussed, open questions and challenges addressed, and future developments outlined. We hope to provide insight for practicing physicians when confronted with basic science articles and encourage investigators to apply "state-of-the-art" technology to their future experiments.
Topics: Anesthesiology; Biomedical Research; Clustered Regularly Interspaced Short Palindromic Repeats; High-Throughput Nucleotide Sequencing; Humans; Induced Pluripotent Stem Cells; Practice Guidelines as Topic; Research Design
PubMed: 32371742
DOI: 10.1213/ANE.0000000000004801 -
Viruses Feb 2020Viruses are evolving at an alarming rate, spreading and inconspicuously adapting to cutting-edge therapies. Therefore, the search for rapid, informative and reliable... (Review)
Review
Viruses are evolving at an alarming rate, spreading and inconspicuously adapting to cutting-edge therapies. Therefore, the search for rapid, informative and reliable diagnostic methods is becoming urgent as ever. Conventional clinical tests (PCR, serology, etc.) are being continually optimized, yet provide very limited data. Could high throughput sequencing (HTS) become the future gold standard in molecular diagnostics of viral infections? Compared to conventional clinical tests, HTS is universal and more precise at profiling pathogens. Nevertheless, it has not yet been widely accepted as a diagnostic tool, owing primarily to its high cost and the complexity of sample preparation and data analysis. Those obstacles must be tackled to integrate HTS into daily clinical practice. For this, three objectives are to be achieved: (1) designing and assessing universal protocols for library preparation, (2) assembling purpose-specific pipelines, and (3) building computational infrastructure to suit the needs and financial abilities of modern healthcare centers. Data harvested with HTS could not only augment diagnostics and help to choose the correct therapy, but also facilitate research in epidemiology, genetics and virology. This information, in turn, could significantly aid clinicians in battling viral infections.
Topics: Clinical Laboratory Techniques; Computational Biology; Fever of Unknown Origin; High-Throughput Nucleotide Sequencing; Humans; Molecular Diagnostic Techniques; Virus Diseases; Viruses
PubMed: 32074965
DOI: 10.3390/v12020211 -
Cold Spring Harbor Perspectives in... Oct 2019High-throughput sequencing methods have revolutionized our ability to catalog the diversity of RNAs and RNA-protein interactions that can exist in our cells. However,... (Review)
Review
High-throughput sequencing methods have revolutionized our ability to catalog the diversity of RNAs and RNA-protein interactions that can exist in our cells. However, the relationship between RNA sequence, structure, and function is enormously complex, demonstrating the need for methods that can provide quantitative thermodynamic and kinetic measurements of macromolecular interaction with RNA, at a scale commensurate with the sequence diversity of RNA. Here, we discuss a class of methods that extend the core functionality of DNA sequencers to enable high-throughput measurements of RNA folding and RNA-protein interactions. Topics discussed include a description of the method and multiple applications to RNA-binding proteins, riboswitch design and engineering, and RNA tertiary structure energetics.
Topics: High-Throughput Nucleotide Sequencing; Kinetics; Nucleic Acid Conformation; RNA; RNA-Binding Proteins; Thermodynamics
PubMed: 30322887
DOI: 10.1101/cshperspect.a032300 -
Archives of Pathology & Laboratory... Jan 2022The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the...
CONTEXT.—
The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature.
OBJECTIVE.—
To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens.
DESIGN.—
We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens.
RESULTS.—
Identification of specimen contamination was validated via in silico and in vitro mixtures, and reference range and reproducibility were established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively applied this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) were predicted to be affected by greater than 5% contamination.
CONCLUSIONS.—
Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.
Topics: High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Pathology, Molecular; Polymorphism, Single Nucleotide; Reproducibility of Results
PubMed: 34015814
DOI: 10.5858/arpa.2020-0679-OA