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. 2024 Nov 27;14(1):29531.
doi: 10.1038/s41598-024-80660-3.

Evaluation of DNA extraction kits for long-read shotgun metagenomics using Oxford Nanopore sequencing for rapid taxonomic and antimicrobial resistance detection

Affiliations

Evaluation of DNA extraction kits for long-read shotgun metagenomics using Oxford Nanopore sequencing for rapid taxonomic and antimicrobial resistance detection

Srinithi Purushothaman et al. Sci Rep. .

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

Keywords: Antimicrobial resistance.; DNA extraction; Long-read; Metagenomics; ONT; Protocol; Taxonomy.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Experimental study design. The green box (left) shows the experimental workflow. The blue box (top right) outlines the composition of the two mock communities: the Zymo Mock Community and the in-house ESKAPE Mock Community. The yellow box (lower right) represents the four different DNA extraction kits used. Gram neg, Gram-negative; Gram pos, Gram-positive. R - antimicrobial resistant strain, and S - antimicrobial sensitive strain. The number of tested samples is shown in the green box (left). For the Zymo Mock Community (n = 12), this includes three technical replicates repeated across four sequencing runs (biological replicates). For the ESKAPE Mock (n = 3), the data represents three sequencing runs without technical replicates. The pooled swab samples (n = 9) include three technical replicates repeated across three sequencing runs (biological replicates).
Fig. 2
Fig. 2
Bioinformatics data analysis workflow. The green boxes show the analysis performed on the raw reads, the blue boxes show the preprocessing steps, and the orange boxes show the de novo assembly and the analysis carried out on the de novo assembled contigs.
Fig. 3
Fig. 3
Sequencing quality of different extraction kits and microbial communities. Quality Control and de novo assembly summary from NanoPlot and Flye for the three sample types from four different DNA extraction kits. a-c represents a double box plot plotted total bases generated (in gigabases) against the read length N50 (in kilobases). d-f Double box plot for the total length of assembled contigs (in megabases) against the number of assembled contigs. Each color represents one DNA extraction kit. BS, Buccal Swab; CC, Cultured Cells; DM, QIAamp DNA Mini; and PF, QIAamp PowerFecal Pro DNA. The number of tested samples is provided in the brackets, which represent the technical and biological replicates of the Zymo Mock Community, the pooled swab samples, and the technical replicates of the ESKAPE Mock.
Fig. 4
Fig. 4
Performance of taxonomy identification. a and b Heatmaps with the mean read counts (log10 scale) from the raw reads for the Zymo Mock Community and the ESKAPE Mock using Kraken2. Kits used were BS, Buccal Swab; CC, Cultured Cells; DM, QIAamp DNA Mini; and PF, QIAamp PowerFecal Pro DNA. The total number of tested samples is provided inside the brackets(x-axis) which represent the technical and biological replicates for the Zymo Mock Community and the technical replicates for the ESKAPE Mock. c and d Assembled contigs alignment to the reference genomes of the Zymo Mock Community and ESKAPE Mock. The x-axis represents the different DNA extraction kits, and the y-axis represents the number of identical bases aligned to the respective reference genomes. The horizontal green line indicates the actual genome size of the respective bacterial species.
Fig. 5
Fig. 5
AMR genes prediction. The figure shows the mean gene length coverage percentage on raw reads (left) and assembled contigs (right) for the ESKAPE Mock sample. Kits used were BS, Buccal Swab; CC, Cultured Cells; DM, QIAamp DNA Mini; and PF, QIAamp PowerFecal Pro DNA. The total number of tested samples is given inside the brackets (x-axis), which represent the technical replicates of the ESKAPE Mock. The respective gene lengths of the detected AMR genes are shown in brackets (y-axis). The white box represents the absence of AMR genes (0% gene length coverage).

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