Genomic DNA was extracted using the Wizard DNA extraction kit (Promega; catalog no. A1125). DNA was quantified using a dsDNA Broad Range fluorometric quantification assay (Invitrogen; catalog no. Q32853). Double-stranded DNA libraries (average, 500 bp) were prepared using the Covaris LC220 for fragmentation and NEBNext Ultra II FS DNA library kit for Illumina with 384-unique indexes (New England Biolabs; catalog no. E6617L). Libraries were sequenced using the HiSeq X10 with 150 bp paired-end chemistry (Illumina).
Dsdna broad range fluorometric quantification assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DsDNA Broad Range fluorometric quantification assay is a laboratory tool used to measure the concentration of double-stranded DNA in a sample. It utilizes a fluorescent dye that binds to DNA, enabling the quantification of DNA present in the sample.
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2 protocols using dsdna broad range fluorometric quantification assay
1
Genomic DNA Extraction and Sequencing
2
Genomic Surveillance of Antimicrobial Resistance
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DNA extraction, Library Preparation, and Sequencing: Genomic DNA was extracted using the Wizard DNA extraction kit (Promega; Wisconsin, USA; Cat. No: A1125). DNA was quantified using a dsDNA Broad Range fluorometric quantification assay (Invitrogen; California, USA; Cat. No: Q32853). Double-stranded DNA libraries (avg. 500 bp) were prepared using the Covaris LC220 for fragmentation, and NEBNext Ultra II FS DNA library kit for Illumina with 384-unique indexes (New England Biolabs, Massachusetts, USA; Cat. No: E6617L). Libraries were sequenced using the HiSeq X10 with 150 bp paired-end chemistry (Illumina, CA, USA).
Genome assembly: Genome assembly was carried out according to the GHRU protocol (https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bpn6mmhe).
Parameters for post-assembly quality checks include the total genome size (between 4584497 bp to 7012008 bp), N50 score (> 25000), contaminant level (< 5%), and number of contigs (< 300). Only Klebsiella genomes that passed all quality checks (K. pneumoniae, n = 134; K. quasipneumoniae, n = 5) were selected for downstream analysis.
Genome assembly: Genome assembly was carried out according to the GHRU protocol (https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bpn6mmhe).
Parameters for post-assembly quality checks include the total genome size (between 4584497 bp to 7012008 bp), N50 score (> 25000), contaminant level (< 5%), and number of contigs (< 300). Only Klebsiella genomes that passed all quality checks (K. pneumoniae, n = 134; K. quasipneumoniae, n = 5) were selected for downstream analysis.
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