The optimal standard protocols for whole-genome sequencing of antibiotic-resistant pathogenic bacteria using third-generation sequencing platforms

다국어 초록 (Multilingual Abstract)

Background The emergence of antibiotic-resistant bacterial pathogens in the environment has been increasing, posing a threat to public health. Next-generation sequencing technology, which is both low cost and large scale, was used to identify antibiotic-resistance genes or toxin genes in these pathogens. Short-read sequencing cannot fully reconstruct bacterial chromosomes and plasmids carrying toxin genes and antibiotic-resistance genes because of their location on the insertion sequences or repeat regions. Third-generation sequencing generated long reads that could cover the repetitive and/or insertion sequences, allowing for complete chromosome and plasmid reconstruction. However, the optimal protocols for whole-genome sequencing of antibiotic-resistance pathogenic bacteria, from DNA extraction to genome assembly, are still being researched.




Objective To develop a pipeline of optimal methods for whole-genome sequencing of bacteria, we compared three commercial DNA extraction kits (column extraction, magnetic bead extraction, and precipitation extraction), two third-generation sequencing platforms (MinION and PacBio), and three assembly methods (flye, unicycler, and unicycler_hybrid). The assembly results were evaluated based on the number of contigs and the detection of anti-microbial-resistance genes.




Results Magnetic bead extraction method generated longer N50 read lengths and greater read length distribution than the other two extraction methods. The Flye assembler in combination with magnetic bead extraction and MinION sequencing provided consistent successful plasmid assembly results and detected all antimicrobial-resistance gene in all datasets.




Conclusion DNA extraction, sequencing platform, and assembly methods can all have an impact on the results of bacterial whole-genome sequencing. Our findings could be a practical protocol for researchers who use third-generation sequencing to perform bacterial whole-genome sequencing by consistently resolving small plasmids carrying antibiotic-resistance genes.

다국어 초록 (Multilingual Abstract)

Background The emergence of antibiotic-resistant bacterial pathogens in the environment has been increasing, posing a threat to public health. Next-generation sequencing technology, which is both low cost and large scale, was used to identify antibiotic-resistance genes or toxin genes in these pathogens. Short-read sequencing cannot fully reconstruct bacterial chromosomes and plasmids carrying toxin genes and antibiotic-resistance genes because of their location on the insertion sequences or repeat regions. Third-generation sequencing generated long reads that could cover the repetitive and/or insertion sequences, allowing for complete chromosome and plasmid reconstruction. However, the optimal protocols for whole-genome sequencing of antibiotic-resistance pathogenic bacteria, from DNA extraction to genome assembly, are still being researched.


Objective To develop a pipeline of optimal methods for whole-genome sequencing of bacteria, we compared three commercial DNA extraction kits (column extraction, magnetic bead extraction, and precipitation extraction), two third-generation sequencing platforms (MinION and PacBio), and three assembly methods (flye, unicycler, and unicycler_hybrid). The assembly results were evaluated based on the number of contigs and the detection of anti-microbial-resistance genes.


Results Magnetic bead extraction method generated longer N50 read lengths and greater read length distribution than the other two extraction methods. The Flye assembler in combination with magnetic bead extraction and MinION sequencing provided consistent successful plasmid assembly results and detected all antimicrobial-resistance gene in all datasets.


Conclusion DNA extraction, sequencing platform, and assembly methods can all have an impact on the results of bacterial whole-genome sequencing. Our findings could be a practical protocol for researchers who use third-generation sequencing to perform bacterial whole-genome sequencing by consistently resolving small plasmids carrying antibiotic-resistance genes.

참고문헌 (Reference)

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