Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples

D’Andreano, Sara; Cuscó, Anna; Francino, Olga

doi:10.1101/2020.02.06.936708

Cited by 12 publications

(10 citation statements)

References 29 publications

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“…[20,21], whereas a very recent study demontrated a correlation between bacterial and fungal populations of the external ear canal and otitis caused by Pseudomonas and Malassezia [21]. Malassezia pachydermatis has been reported as the dominant fungi in the ear skin of healthy dogs, while dogs with clinical otitis exhibited an overgrowth of M. globosa and M. sympodialis [22].…”

Section: Introductionmentioning

confidence: 99%

Cerumen microbial community shifts between healthy and otitis affected dogs

et al. 2020

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Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.

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Section: Introductionmentioning

confidence: 99%

Cerumen microbial community shifts between healthy and otitis affected dogs

et al. 2020

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“…Although a mature pipeline comprising ITS amplification, IIllumina sequencing and data analysis has been established [19], several studies reported biases from the sequencing technology used and from unevenly amplified fungal marker regions [20][21][22]. Recently, novel strategies exploiting long-range amplification and long-read sequencing have been developed to improve these classifications [23,24]. In addition, whole genome shotgun sequencing and rapidly expanding genome databases allow mapping of newly generated DNA sequences directly to the database.…”

Section: Introductionmentioning

confidence: 99%

Inferring species compositions of complex fungal communities from long- and short-read sequence data

Irinyi

Hoang

et al. 2021

Preprint

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Background: The kingdom fungi is crucial for life on earth and is highly diverse. Yet fungi are challenging to characterize. They can be difficult to culture and may be morphologically indistinct in culture. They can have complex genomes of over 1 Gb in size and are still underrepresented in whole genome sequence databases. Overall their description and analysis lags far behind other microbes such as bacteria. At the same time, classification of species via high throughput sequencing without prior purification is increasingly becoming the norm for pathogen detection, microbiome studies, and environmental monitoring. However, standardized procedures for characterizing unknown fungi from complex sequencing data have not yet been established. Results: We compared different metagenomics sequencing and analysis strategies for the identification of fungal species. Using two fungal mock communities of 44 phylogenetically diverse species, we compared species classification and community composition analysis pipelines using shotgun metagenomics and amplicon sequencing data generated from both short and long read sequencing technologies. We show that regardless of the sequencing methodology used, the highest accuracy of species identification was achieved by sequence alignment against a fungi-specific database. During the assessment of classification algorithms, we found that applying cut-offs to the query coverage of each read or contig significantly improved the classification accuracy and community composition analysis without significant data loss. Conclusion: Overall, our study expands the toolkit for identifying fungi by improving sequence-based fungal classification, and provides a practical guide for the design of metagenomics analyses.

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“…Nanopore targeted sequencing (NTS), which amplified marker genes and used nanopore sequencing platform to sequence the amplified marker genes, were developed for investigation of bacterial or fungal infection. However, previous NTS was only based on a single marker gene, such as the 16s rRNA gene targeted sequencing that has been reported to identify bacterial infection in individual clinical case reports (23, 24) , and fungal ribosomal operon (18S-ITS1-5.8S-ITS2-28S) has been used to detect the fungal community associated with external otitis in dogs (25) . Simultaneous broad-spectrum detections of bacteria and fungi, and high-sensitivity detection of critical clinical pathogens in clinical samples by a single test based on NTS have not been developed.…”

Section: Introductionmentioning

confidence: 99%

Same-Day Simultaneous Diagnosis of Bacterial and Fungal Infections in Clinical Practice by Nanopore Targeted Sequencing

Wang

et al. 2020

Preprint

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BACKGROUND: As approximately 19% of global deaths are attributable to infectious diseases, early diagnosis of infection is very important to reduce mortality. Traditional infection detection strategies have limited sensitivity, detection range, and turnaround times; a detection technology that can simultaneously detect bacterial and fungal infections within 24 h is urgently need in clinical settings. METHODS: We developed nanopore targeted sequencing (NTS) for same-day simultaneous diagnosis of fungal and bacterial infections. NTS was developed by amplification of 16s rRNA gene (for bacteria), IST1/2 gene (for fungal), and rpoB (for Mycobacterium spp.) using multiple primers, and sequenced by a real-time nanopore sequencing platform. An in-house bioinformatic analyze pipeline was used to diagnose the infectious pathogens by mapping the sequencing results with the constructed databases. RESULTS: Comparison of 1312 specimens from 1257 patients using NTS and culture method; NTS detected pathogens in 58.71% of specimens from patients, compared to 22.09% detected using the culture method. NTS showed significantly higher sensitivity than culture methods for many pathogens. Importantly, a turnaround time of <24 h for all specimens, and a pre-report within 6 h in emergency cases was possible in clinical practice. Modification of antibiotic therapy and maintenance of original anti-infection regimens in 51.52% (17/33) and 36.36% (12/33) of patients was in accordance with NTS results, and quantitative monitoring of clinical treatment effects was evaluated in four patients by continuous NTS tests. CONCLUSIONS: Application of NTS in clinically detected pathogens can improve targeted antibiotic treatment and therapeutic monitoring.

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Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples

Cited by 12 publications

References 29 publications

Cerumen microbial community shifts between healthy and otitis affected dogs

Cerumen microbial community shifts between healthy and otitis affected dogs

Inferring species compositions of complex fungal communities from long- and short-read sequence data

Same-Day Simultaneous Diagnosis of Bacterial and Fungal Infections in Clinical Practice by Nanopore Targeted Sequencing

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