Rapid whole genome characterization of antimicrobial-resistant pathogens using long-read sequencing to identify potential healthcare transmission DOI Creative Commons

Chin-Ting Wu,

William C. Shropshire, Micah M. Bhatti

et al.

Infection Control and Hospital Epidemiology, Journal Year: 2024, Volume and Issue: 46(2), P. 129 - 135

Published: Dec. 27, 2024

Whole genome sequencing (WGS) can help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard short-read, Illumina-based WGS is labor and time intensive. Given recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource approach providing accurate WGS-pathogen comparison within frame allowing for infection prevention control (IPC) interventions. was prospectively performed on at increased risk potential healthcare using ONT MinION sequencer with R10.4.1 flow cells Dorado basecaller. Potential assessed via Ridom SeqSphere+ core multilocus sequence typing MINTyper reference-based single nucleotide polymorphisms previously published cutoff values. The accuracy our pipeline determined relative Illumina. Over six-month period, 242 bacterial isolates from 216 patients were sequenced by operator. Compared Illumina standard, achieved mean identity score Q60 assembled genomes, even coverage rate as 40×. initiating DNA extraction complete analysis 2 days (IQR 2-3.25 days). We identified five clusters comprising 21 (8.7% strains). Integrating epidemiological data, >70% (15/21) putative cluster originated links. Via stand-alone pipeline, detected potentially transmitted HAI rapidly accurately, aligning closely data. Our low-resource method has assist IPC efforts.

Language: Английский

Benchmarking reveals superiority of deep learning variant callers on bacterial nanopore sequence data DOI Creative Commons
Michael B. Hall, Ryan R. Wick, Louise M. Judd

et al.

eLife, Journal Year: 2024, Volume and Issue: 13

Published: May 20, 2024

Variant calling is fundamental in bacterial genomics, underpinning the identification of disease transmission clusters, construction phylogenetic trees, and antimicrobial resistance detection. This study presents a comprehensive benchmarking variant accuracy genomes using Oxford Nanopore Technologies (ONT) sequencing data. We evaluated three ONT basecalling models both simplex (single-strand) duplex (dual-strand) read types across 14 diverse species. Our findings reveal that deep learning-based callers, particularly Clair3 DeepVariant, significantly outperform traditional methods even exceed Illumina sequencing, especially when applied to ONT’s super-high model. superior performance attributed its ability overcome Illumina’s errors, which often arise from difficulties aligning reads repetitive variant-dense genomic regions. Moreover, use high-performing callers with data mitigates errors homopolymers. also investigated impact depth on calling, demonstrating 10× super-accuracy can achieve precision recall comparable to, or better than, full-depth sequencing. These results underscore potential combined advanced algorithms, replace short-read resource-limited settings.

Language: Английский

Citations

7
Characteristics and filtering of low-frequency artificial short deletion variations based on nanopore sequencing DOI Creative Commons
Fuqiang Ye, Juanjuan Zhu, Xiaomin Zhang

et al.

GigaScience, Journal Year: 2025, Volume and Issue: 14

Published: Jan. 1, 2025

Nanopore sequencing is characterized by high portability and long reads, albeit accompanied systematic errors causing short deletions. Few tools can filter low-frequency artificial deletions, especially in single samples. To solve this problem, we first synthesized or purchased 17 DNA/RNA standards for nanopore with R9 R10 flowcells to obtain benchmarking datasets. False-positive (FP) deletions were prevalent (75.86%-96.26%), while the majority (62.07%-79.68%) located homopolymeric regions. The 10-mer base-quality scores (Q scores) speeds flanking FP marginally differed from true-positive (TP) We thus investigated raw current signals after normalizing them length. found more significant differences between reads without Indexes including MRPP A (Multiple Response Permutation Procedure, statistic A), accumulative difference of normalized signals, Q score tested power distinguishing TP outperformed other indexes regions achieved highest accuracy 76.73% challenging 1-base When depth was low, performed better than A. developed Delter (Deletion filter) samples, which removed 60.98% 100% real Low-frequency deletion variations, most could be effectively filtered using according employed strategies.

Language: Английский

Citations

0
Transcriptomics in the era of long-read sequencing DOI Creative Commons
Carolina Monzó, Tianyuan Liu, Ana Conesa

et al.

Nature Reviews Genetics, Journal Year: 2025, Volume and Issue: unknown

Published: March 28, 2025

Language: Английский

Citations

0
Custom barcoded primers for influenza A nanopore sequencing: enhanced performance with reduced preparation time DOI Creative Commons
Iryna V. Goraichuk, David L. Suarez

Frontiers in Cellular and Infection Microbiology, Journal Year: 2025, Volume and Issue: 15

Published: April 15, 2025

Highly pathogenic avian influenza is endemic and widespread in wild birds causing major outbreaks poultry worldwide U.S. dairy cows, with several recent human cases, highlighting the need for reliable rapid sequencing to track mutations that may facilitate viral replication different hosts. SNP analysis a useful molecular epidemiology tool outbreaks, but it requires accurate whole-genome (WGS) sufficient read depth across all eight segments. In outbreak situations, where timely data critical controlling spread of virus, reducing preparation time while maintaining high-quality standards particularly important. this study, we optimized custom barcoded primer strategy A on nanopore platform, combining high performance Native Barcoding Kit prompt Rapid Kit. Custom primers were designed perform barcode attachment during RT-PCR amplification, eliminating separate barcoding clean-up steps, thus library time. We compared method kits terms quality, depth, output. The results show provided comparable by 2.3X kit being only 15 minutes longer than better sequencing. Additionally, was evaluated variety clinical sample types. This approach offers promising solution sequencing, providing both throughput efficiency, which significantly improves time-to-result turnaround, making more accessible real-time surveillance.

Language: Английский

Citations

0
Artificial intelligence in variant calling: a review DOI Creative Commons
Omar Abdelwahab, Davoud Torkamaneh

Frontiers in Bioinformatics, Journal Year: 2025, Volume and Issue: 5

Published: April 23, 2025

Artificial intelligence (AI) has revolutionized numerous fields, including genomics, where it significantly impacted variant calling, a crucial process in genomic analysis. Variant calling involves the detection of genetic variants such as single nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and structural from high-throughput sequencing data. Traditionally, statistical approaches have dominated this task, but advent AI led to development sophisticated tools that promise higher accuracy, efficiency, scalability. This review explores state-of-the-art AI-based tools, DeepVariant, DNAscope, DeepTrio, Clair, Clairvoyante, Medaka, HELLO. We discuss their underlying methodologies, strengths, limitations, performance metrics across different technologies, alongside computational requirements, focusing primarily on SNP InDel detection. By comparing these AI-driven techniques with conventional methods, we highlight transformative advancements introduced its potential further enhance research.

Language: Английский

Citations

0
Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies DOI Creative Commons
George Bouras, Ghais Houtak, Ryan R. Wick

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 13, 2023

Abstract Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- long-reads) assembly approaches. Complete allow a deeper understanding evolution genomic variation beyond single nucleotide variants (SNVs). They also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids missed or misassembled by algorithms. Here, we present Hybracter allows fast, automatic, scalable recovery near-perfect first approach. can be run either as assembler only assembler. We compared to existing automated tools diverse panel samples varying levels with manually curated ground truth reference genomes. demonstrate is more accurate faster than gold standard Unicycler. show long-reads most comparable methods accurately recovering plasmids. Data Summary developed Python Snakemake command-line software tool Linux MacOS systems. freely available under an MIT License on GitHub ( https://github.com/gbouras13/hybracter ) documentation at Read Docs https://hybracter.readthedocs.io/en/latest/ ). install via PyPI https://pypi.org/project/hybracter/ Bioconda https://anaconda.org/bioconda/hybracter A Docker/Singularity container https://quay.io/repository/gbouras13/hybracter . All code used benchmark Hybracter, including genomes, publicly https://github.com/gbouras13/hybracter_benchmarking released DOI https://zenodo.org/doi/10.5281/zenodo.10910108 Zenodo. The subsampled FASTQ files benchmarking Zenodo https://doi.org/10.5281/zenodo.10906937 super simplex ATCC reads sequenced part this study found BioProject PRJNA1042815. Hall et al. fast duplex read (prior subsampling) SRA PRJNA1087001. raw Lermaniaux PRJNA1020811. Staphylococcus aureus JKD6159 PRJNA50759. Mycobacterium tuberculosis H37R2 PRJNA836783. list BioSample accession numbers each benchmarked sample Supplementary Table 1. output Pypolca outputs https://zenodo.org/doi/10.5281/zenodo.10072192 Impact Statement genome routine vital genomics, especially identification mobile genetic elements As becomes cheaper, easier access accurate, crucial. With new widely-used both only. Additionally, it solves problems assemblers struggling plasmid from performing par methods. natively exploit parallelisation high-performance computing (HPC) clusters cloud-based environments, enabling users assemble hundreds thousands one line code. source GitHub, PyPi.

Language: Английский

Citations

7
Rapid whole genome characterization of antimicrobial-resistant pathogens using long-read sequencing to identify potential healthcare transmission DOI Creative Commons

Chin-Ting Wu,

William C. Shropshire, Micah M. Bhatti

et al.

Infection Control and Hospital Epidemiology, Journal Year: 2024, Volume and Issue: 46(2), P. 129 - 135

Published: Dec. 27, 2024

Whole genome sequencing (WGS) can help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard short-read, Illumina-based WGS is labor and time intensive. Given recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource approach providing accurate WGS-pathogen comparison within frame allowing for infection prevention control (IPC) interventions. was prospectively performed on at increased risk potential healthcare using ONT MinION sequencer with R10.4.1 flow cells Dorado basecaller. Potential assessed via Ridom SeqSphere+ core multilocus sequence typing MINTyper reference-based single nucleotide polymorphisms previously published cutoff values. The accuracy our pipeline determined relative Illumina. Over six-month period, 242 bacterial isolates from 216 patients were sequenced by operator. Compared Illumina standard, achieved mean identity score Q60 assembled genomes, even coverage rate as 40×. initiating DNA extraction complete analysis 2 days (IQR 2-3.25 days). We identified five clusters comprising 21 (8.7% strains). Integrating epidemiological data, >70% (15/21) putative cluster originated links. Via stand-alone pipeline, detected potentially transmitted HAI rapidly accurately, aligning closely data. Our low-resource method has assist IPC efforts.

Language: Английский

Citations

0