Comparison Analysis of Different DNA Extraction Methods on Suitability for Long-Read Metagenomic Nanopore Sequencing

Zhang, Lei; Chen, Ting; Wang, Ye; Zhang, Shengwei; Lv, Qingyu; Kong, Decong; Jiang, Hua; Zheng, Yinan; Ren, Yilong; Huang, Wenhua; Liu, Peng; Jiang, Yongqiang

doi:10.3389/fcimb.2022.919903

Cited by 11 publications

(8 citation statements)

References 29 publications

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“…C. auris genomic DNA was extracted using an enzymatic lysis method as previously described. 14 Whole-genome sequencing was conducted using both the Illumina and Nanopore platforms as described in the Supplementary Methods . The ITS and D1/D2 (region of the large subunit (28S) of the ribosomal DNA) sequences of C. auris and previous reported isolates were aligned using ClustalW2 and a maximum likelihood phylogenetic tree was generated using IQ-TREE with 1000 bootstrap repeats.…”

Section: Methodsmentioning

confidence: 99%

A Candidemia Case Caused by a Novel Drug-Resistant Candida auris with the Y132F Mutation in Erg11 in Mainland China

Xu¹,

Zhang²,

Han³

et al. 2023

IDR

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Background Candida auris is a pathogen first found in external ear canal, becoming a major threat to global health. Here, we describe a candidemia case caused by a novel drug-resistant Candida auris strain. Case Presentation An 80-year-old patient, with multiple serious medical conditions, was suffered from candidemia caused by Candida auris , died 9 days after admission in our hospital. Phylogenetic analysis indicates that this C. auris isolate (designated BJCA003) belongs to the South Asian clade, carries the Y132F mutation in the protein Erg11. And antibiotic susceptibility test indicated that BJCA003 is resistant to fluconazole and amphotericin B, not susceptible to caspofungin. In addition, this strain has multiple colony and cellular morphologies under different culture conditions. Conclusion Strain BJCA003 is a novel drug resistant C. auris strain in mainland China, the Y132F mutation in Erg11 may attribute to fluconazole-resistance, alarming that we still face more challenges about C. auris.

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Section: Methodsmentioning

confidence: 99%

A Candidemia Case Caused by a Novel Drug-Resistant Candida auris with the Y132F Mutation in Erg11 in Mainland China

Xu¹,

Zhang²,

Han³

et al. 2023

IDR

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“…Implementing methods that selectively lyse host cells and/or separate specific organisms from clinical samples [ 99 , 100 ]…”

Section: Figurementioning

confidence: 99%

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

Kan,

Tsang,

Pei

et al. 2024

IJMS

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Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives.

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“…The QIAamp BiOstic Bacteremia DNA Kit, DNeasy Blood & Tissue Kit, and PowerFood Microbial Kit have been the most used kits for the isolation of DNA from urine samples. Particularly, the BiOstic Bacteremia DNA Kit and the Blood & Tissue Kit were found to preserve the bacterial diversity of the sample while yielding a large amount of good-quality DNA for downstream sequencing (21,24,25).…”

Section: Introductionmentioning

confidence: 99%

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

Bellankimath,

Chapagain,

Branders

et al. 2024

Preprint

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Purpose: Urinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. Methods: This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 10^3 and 10^5 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. Results: The Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 10^5 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 10^3 CFU/mL. Conclusion: This study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

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Comparison Analysis of Different DNA Extraction Methods on Suitability for Long-Read Metagenomic Nanopore Sequencing

Cited by 11 publications

References 29 publications

A Candidemia Case Caused by a Novel Drug-Resistant Candida auris with the Y132F Mutation in Erg11 in Mainland China

A Candidemia Case Caused by a Novel Drug-Resistant Candida auris with the Y132F Mutation in Erg11 in Mainland China

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

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