Genomic DNA was extracted using a Qiagen DNeasy UltraClean Microbial DNA Isolation Kit. A Nextera XT DNA Library Prep Kit was used to prepare barcoded libraries, which were sequenced using an Illumina MiSeq v3 sequencing kit. Multilocus sequence typing and characterization of antibiotic-resistance genes was performed using SRST2 (14 (link)). To generate accurate reference genomes for isolate series, we also conducted long-read sequencing of strains A1, B1, and C1 on a MinION (Oxford Nanopore Technologies) after library preparation with the Rapid Barcoding Sequencing Kit. Reads were basecalled using MinKnow and Epi2ME (Metrichor), and hybrid de novo assembly of Nanopore and Illumina reads was performed using SPAdes v3.10.1 (6 (link)). The genome was annotated using Prokka v1.12 (15 (link)), and mobile genetic elements and prophage regions were identified by IslandViewer 4 (16 (link)) and PHASTER (17 (link)), respectively. We used Snippy software (Snippy v3, https://github.com/tseemann/snippy ) to identify core genome concatenated SNPs.
Protocol detail
Genomic Characterization of Bacterial Isolates
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DNeasy UltraClean Microbial DNA Isolation Kit
Nextera XT DNA Library Prep Kit
MiSeq v3 sequencing kit
MinION
Antibiotic resistance
Genes
Genome
Hybrid
Isolation
Library
Mobile genetic elements
Prophage
Snps
Strains
Corresponding Organization :
Other organizations : Pediatrics and Genetics, Columbia University Irving Medical Center, Yale University
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Variable analysis
independent variables
- Library preparation using Nextera XT DNA Library Prep Kit
- Sequencing using Illumina MiSeq v3 sequencing kit
- Long-read sequencing of strains A1, B1, and C1 on a MinION (Oxford Nanopore Technologies) after library preparation with the Rapid Barcoding Sequencing Kit
- Multilocus sequence typing
- Characterization of antibiotic-resistance genes
- Generation of accurate reference genomes for isolate series
- Identification of mobile genetic elements and prophage regions
- Identification of core genome concatenated SNPs
- DNA extraction using Qiagen DNeasy UltraClean Microbial DNA Isolation Kit
- Basecalling of Nanopore reads using MinKnow and Epi2ME (Metrichor)
- Hybrid de novo assembly of Nanopore and Illumina reads using SPAdes v3.10.1
- Genome annotation using Prokka v1.12
- Identification of mobile genetic elements and prophage regions using IslandViewer 4 and PHASTER
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