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Comparison of three commercial DNA extraction kits and assemblers for AMR determinant detection in Pseudomonas
Anurag Kumar Bari1, Basil Britto Xavier1, Tim Severs2
1Department of Medical Microbiology and Infection Prevention, Antimicrobial Resistance, Genomics and Epidemiology (AGE) Research Group, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Background:
Pseudomonas aeruginosa and Enterobacter cloacae, both members of the ESKAPE group, are multidrug-resistant pathogens that pose significant challenges in clinical care. The high GC-content (∼67 %) of the P. aeruginosa genome complicates DNA extraction and long-read sequencing, with downstream effects on genome assembly and analyses. We compared three commercial extraction kits for isolating high-quality DNA suitable for Oxford Nanopore sequencing from clinical isolates of P. aeruginosa and E. cloacae.
Methods:
Genomic DNA was extracted from P. aeruginosa (n = 63; 3 × 21) and E. cloacae (n = 96; 3 × 32) clinical isolates using the MagAttract HMW DNA Kit (Qiagen), the DNeasy UltraClean Microbial Kit (Qiagen), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (ThermoFisher). DNA quantity/quality was assessed by spectrophotometry (NanoDrop, Thermo Fisher Scientific), fluorometry (Qubit, ThermoFisher), and capillary electrophoresis (TapeStation 2200, Agilent). Libraries were prepared and sequenced using Oxford Nanopore Technologies platforms. Assemblies were generated with Unicycler (v.0.5.1) and Flye (v.2.9.6); quality was assessed with QUAST (v.5.3). Genome completeness was evaluated by CheckM (v.1.1.6). Antimicrobial resistance determinants were identified with AMRFinderPlus (v.4.1.19).
Results:
DNeasy yielded up to 4.7× higher DNA and ∼ 50 % higher sequencing output than MagAttract, while MagAttract produced higher DNA integrity and more contiguous assemblies. The choice of assembly had a greater impact on the detection of AMR determinants than the extraction method alone. Across workflows, Flye outperformed Unicycler, increasing detection by 2-14 percentage points. The best-performing combination (DNeasy + Flye) achieved 95.2 % AMR determinants, compared to 67.8 % for MagMAX + Unicycler, with the most difference (37.5 %) in efflux pump genes.
Conclusions:
This systematic comparison highlights trade-offs between DNA yield, integrity, and downstream assembly performance, demonstrating that assembler choice critically impacts the detection of AMR determinants. These findings provide practical guidance for optimizing long-read-based sequencing workflows to support AMR surveillance and genomic epidemiology.
Insights
Choosing the right DNA extraction kit and genome assembler is crucial for accurately detecting antimicrobial resistance (AMR) determinants in pathogens like Pseudomonas aeruginosa and Enterobacter cloacae using long-read sequencing.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- Multidrug-resistant pathogens Pseudomonas aeruginosa and Enterobacter cloacae present significant clinical challenges.
- High GC-content in P. aeruginosa complicates DNA extraction and sequencing, impacting genome assembly.
- Effective DNA extraction is vital for accurate genomic analysis of these ESKAPE pathogens.
Purpose of the Study:
- To compare the performance of three commercial DNA extraction kits for long-read sequencing of P. aeruginosa and E. cloacae.
- To evaluate the impact of DNA extraction methods and genome assembly tools on antimicrobial resistance (AMR) determinant detection.
- To provide practical guidance for optimizing long-read sequencing workflows for AMR surveillance.
Main Methods:
- Compared three commercial kits (MagAttract, DNeasy, MagMAX) for DNA extraction from clinical isolates.
- Assessed DNA quantity, quality, and integrity using spectrophotometry, fluorometry, and capillary electrophoresis.
- Sequenced DNA using Oxford Nanopore Technologies and assembled genomes with Unicycler and Flye.
Main Results:
- DNeasy kit yielded higher DNA quantity and sequencing output compared to MagAttract.
- MagAttract kit provided higher DNA integrity and more contiguous genome assemblies.
- The Flye assembler significantly improved AMR determinant detection compared to Unicycler, with the DNeasy + Flye combination achieving the highest detection rate.
Conclusions:
- DNA extraction method and assembler choice significantly impact AMR determinant detection.
- Optimizing DNA extraction and assembly is critical for accurate AMR surveillance using long-read sequencing.
- Findings offer practical insights for enhancing genomic epidemiology studies of resistant bacteria.
