Filtering out the noise: metagenomic classifiers optimize ancient DNA mapping DOI Creative Commons
Shyamsundar Ravishankar, Vilma Pérez, Roberta Davidson

et al.

Briefings in Bioinformatics, Journal Year: 2024, Volume and Issue: 26(1)

Published: Nov. 22, 2024

Abstract Contamination with exogenous DNA presents a significant challenge in ancient (aDNA) studies of single organisms. Failure to address contamination from microbes, reagents, and present-day sources can impact the interpretation results. Although field laboratory protocols exist limit contamination, there is still need accurately distinguish between endogenous data computationally. Here, we propose workflow reduce based on metagenomic classifier. Unlike previous methods that relied exclusively sequencing reads mapping specificity reference genome remove contaminating reads, our approach uses Kraken2-based filtering before genome. Using both simulated empirical shotgun aDNA data, show this simple efficient method be used wide range computational environments—including personal machines. We strategies build specific databases profile take into consideration available resources prior knowledge about target taxa likely contaminants. Our significantly reduces overall required during process total runtime by up ~94%. The most impacts are observed low samples. Importantly, contaminants would map filtered out using strategy, reducing false positive alignments. also results negligible loss no measurable downstream population genetics analyses.

Language: Английский

Unveiling microbial diversity: harnessing long-read sequencing technology DOI
Daniel Paiva Agustinho, Yilei Fu, Vipin K. Menon

et al.

Nature Methods, Journal Year: 2024, Volume and Issue: 21(6), P. 954 - 966

Published: April 30, 2024

Language: Английский

Citations

29
CArP - CApture-based sequencing for Pathogen surveillance in complex matrices DOI Creative Commons
Paul B. Stege, Frank Harders, Alex Bossers

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 20, 2025

Abstract This study highlights the development and application of a novel capture-based long-read sequencing approach, CArP, designed to benefit pathogen surveillance. combines targeted gene enrichment with contextual insights sequencing, providing highly efficient flexible approach for detection. In this initial version, antimicrobial resistance (AMR) genes are utilized as marker that improve sensitivity. allowed detection bacteria in broiler caecal content samples carried critical antibiotics, including macrolides quinolones. These specific AMR would have remained undetected when traditional metagenomic shotgun been applied, it failed detect these therefore putative pathogens. demonstrates potential characterize pathogens more effectively compared strategies. The modular design CArP furthermore expansion additional genes, such viral or fungal markers, can enable broader coverage. Taken together, allows advance contribute global efforts pandemic preparedness subsequent response.

Language: Английский

Citations

0
Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses DOI Creative Commons
Sarah Buddle,

Leysa Forrest,

Naomi Akinsuyi

et al.

Genome Medicine, Journal Year: 2024, Volume and Issue: 16(1)

Published: Sept. 9, 2024

Language: Английский

Citations

3
Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses DOI Creative Commons
Sarah Buddle,

Leysa Forrest,

Naomi Akinsuyi

et al.

medRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 29, 2024

Abstract Background Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, barriers to broader take-up include need high depths, long turnaround times, limited sensitivity. Newer metagenomics protocols Oxford Nanopore Technologies (ONT) allow acquisition analysis data real time, potentially reducing high-volume enabling point-of-care testing. Furthermore, targeted approaches that selectively amplify known pathogens could improve Methods We evaluated viruses with readily available untargeted metagenomic workflows using ONT, an Illumina-based enrichment Twist Biosciences Comprehensive Viral Research Panel (VRP), which targets 3153 viruses. tested samples consisting dilution series six-virus mock community human DNA/RNA background, designed resemble clinical specimens low microbial abundance host content. Protocols were retain transcriptome, since this help confirm absence infectious agents. further compared performance commonly used taxonomic classifiers. Results Capture VRP increased sensitivity by at least 10-100-fold over sequencing, making it suitable viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed setting detect organisms. While ONT had good (60,000 gc/ml), lower (600-6,000 longer more costly runs would required achieve sensitivities comparable protocol. Untargeted provided better specificity than sequencing. application robust thresholds standardized results between Host gene expression optimal possible both ONT. Conclusions has potential become standard-of-care diagnostics tool discovery emerging capture have different advantages respect sensitivity, specificity, time cost, method will depend context.

Language: Английский

Citations

2
Oxford Nanopore Technology-Based Identification of an Acanthamoeba castellanii Endosymbiosis in Microbial Keratitis DOI Creative Commons
Sebastian Scharf,

Lennart Friedrichs,

Robert M. Bock

et al.

Microorganisms, Journal Year: 2024, Volume and Issue: 12(11), P. 2292 - 2292

Published: Nov. 12, 2024

(1) Background: Microbial keratitis is a serious eye infection that carries significant risk of vision loss.

Language: Английский

Citations

2
MetaAll: integrative bioinformatics workflow for analysing clinical metagenomic data DOI Creative Commons
Martin Bosilj, Alen Suljič, Samo Zakotnik

et al.

Briefings in Bioinformatics, Journal Year: 2024, Volume and Issue: 25(6)

Published: Sept. 23, 2024

Abstract Over the past decade, there have been many improvements in field of metagenomics, including sequencing technologies, advances bioinformatics and development reference databases, but a one-size-fits-all pipeline does not yet seem achievable. In this study, we address part analysis by combining three methods into three-step workflow that increases sensitivity specificity clinical metagenomics improves pathogen detection. The individual tools are combined user-friendly suitable for analysing short paired-end (PE) long reads from datasets—MetaAll. To demonstrate applicability developed workflow, four complicated cases with different disease presentations multiple samples collected biological sites as well CAMI Clinical detection challenge dataset were used. MetaAll was able to identify putative pathogens all one case. case, however, traditional microbiological diagnostics also unsuccessful. addition, co-infection Haemophilus influenzae Human rhinovirus C54 detected case 1 SARS-Cov-2 Influenza A virus (FluA) subtype H3N2 3. 2, which conventional could find pathogen, mNGS pointed Klebsiella pneumoniae suspected pathogen. Finally, study demonstrated importance read classification, contig validation targeted mapping more reliable infectious agents metagenome samples.

Language: Английский

Citations

2
Evaluation of DNA extraction kits for long-read shotgun metagenomics using Oxford Nanopore sequencing for rapid taxonomic and antimicrobial resistance detection DOI Creative Commons
Srinithi Purushothaman, Marco Meola, Tim Roloff

et al.

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: Nov. 27, 2024

Abstract During a bacterial infection or colonization, the detection of antimicrobial resistance (AMR) is critical, but slow due to culture-based approaches for clinical and screening samples. Culture-based phenotypic AMR confirmation require up 72 hours (h) even weeks slow-growing bacteria. Direct shotgun metagenomics by long-read sequencing using Oxford Nanopore Technologies (ONT) may reduce time species gene identification. However, swabs complex range Gram-negative -positive bacteria, diverse genes, host DNA present in Therefore, extraction critical initial step. We aimed compare performance different protocols ONT applications reliably identify genes metagenomic approach. included three sample types: ZymoBIOMICS Microbial Community Standard, an in-house mock community ESKAPE pathogens including Enterococcus faecium , Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa Escherichia coli ( Mock ) anonymized swab processed all types with four kits utilizing lysis (enzymatic vs. mechanical) purification (spin-column magnetic beads) methods. used from Qiagen (QIAamp Mini QIAamp PowerFecal Pro DNA) Promega (Maxwell RSC Cultured Cells Maxwell Buccal Swab DNA). After extraction, samples were subject Rapid Barcoding Kit (RBK004) library preparation followed on GridION R9.4.1 flow cells. The fast5 files base called fastq Guppy High Accuracy (HAC) mode inbuilt MinKNOW software. Raw read quality was assessed NanoPlot human reads removed Minimap2 alignment against Hg38 genome. Taxonomy identification performed raw Kraken2 assembled contigs Minimap2. identified CARD database both contigs. Zymo (8/8) (6/6) kit (chemical mechanical lysis) at assembly levels. Enzymatic retrieved fewer aligned bases Gram-positive level compared lysis. detected maximum median 1.9 h sequencing. Long-read turnaround genes. Currently, along captured best taxonomy our specific use case.

Language: Английский

Citations

1
The Naïve Bayes Classifier ++ for Metagenomic Taxonomic Classification—Query Evaluation DOI Creative Commons
Honglei Duan,

Gavin Hearne,

Robi Polikar

et al.

Bioinformatics, Journal Year: 2024, Volume and Issue: 41(1)

Published: Dec. 19, 2024

Abstract Motivation This study examines the query performance of NBC++ (Incremental Naive Bayes Classifier) program for variations in canonicality, k-mer size, databases, and input sample data size. We demonstrate that both Kraken2 are influenced by database depth, with macro measures improving as depth increases. However, fully capturing diversity life, especially viruses, remains a challenge. Results can competitively profile superkingdom content metagenomic samples using small training database. spends less time use fraction memory than but at cost long querying time. Major enhancements include accommodating canonical storage (leading to significant savings) adaptable optimized allocation accelerates analysis enables software be run on nearly any system. Additionally, output now includes log-likelihood values each genome, providing users valuable confidence information. Availability implementation Source code Dockerfile available http://github.com/EESI/Naive_Bayes.

Language: Английский

Citations

1
OCTOPUS: Disk-based, Multiplatform, Mobile-friendly Metagenomics Classifier DOI Creative Commons
Simone Marini, Alexander Barquero,

Anisha Ashok Wadhwani

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 15, 2024

Abstract Portable genomic sequencers such as Oxford Nanopore’s MinION enable real-time applications in clinical and environmental health. However, there is a bottleneck the downstream analytics when bioinformatics pipelines are unavailable, e.g., cloud processing unreachable due to absence of Internet connection, or only low-end computing devices can be carried on site. Here we present platform-friendly software for portable metagenomic analysis Nanopore data, Oligomer-based Classifier Taxonomic Operational Pan-genome Units via Singletons (OCTOPUS). OCTOPUS written Java, reimplements several features popular Kraken2 KrakenUniq software, with original components improving metagenomics classification incomplete/sampled reference databases, making it ideal running smartphones tablets. obtains sensitivity precision comparable Kraken2, while dramatically decreasing (4-to 16-fold) false positive rate, yielding high correlation real-word data. available along customized databases at https://github.com/DataIntellSystLab/OCTOPUS https://github.com/Ruiz-HCI-Lab/OctopusMobile .

Language: Английский

Citations

0
Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms DOI Creative Commons
Lea Schäfer, Johannes A. Jehle, Regina Kleespies

et al.

Journal of Invertebrate Pathology, Journal Year: 2024, Volume and Issue: 205, P. 108121 - 108121

Published: May 4, 2024

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of trees and poses health risks to humans due the urticating setae later instar larvae. For this reason, it difficult rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets early larval instars based primarily on commercial preparations Bacillus thuringiensis ssp. kurstaki (Btk). To test entomopathogenic potential other spore-forming bacteria, user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by bud dipping, (ii) first through feeding exposure (iii) takes into account their group-feeding behavior. A negligible mortality in untreated proved functionality newly established system. Whereas Btk HD-1 strain used as standard confirmed being highly efficient, B. wiedmannii ineffective killing Larvae, which died during infection experiment, were further subjected Nanopore sequencing metagenomic approach entomopathogen detection. It corroborated not able infect establish OPM, but identified insect pathogenic species from genera Serratia Pseudomonas.

Language: Английский

Citations

0