From each specimen, at least three primarily identified E. coli isolates were sequenced. The E. coli strains were selected based on their different Microbact 24E and morphological profiles. DNA was extracted using the Wizard genomic extraction kit (Promega) per the manufacturer’s protocol, and then they were library prepared with the NEBNext Ultra II FS DNA library kit and whole genome sequenced on the Illumina platform at the Wellcome Sanger Institute. Raw read quality control was carried out using FastQC (Babraham Bioinformatics, Babraham Institute, Cambridge, United Kingdom), and quality reports were aggregated using MultiQC.34 (link) Reads were assembled using the SPAdes assembler, and assembly quality was determined using the Quality Assessment Tool for Genome Assemblies (QUAST)35 (link) and CheckM.36 (link) Reads were also assigned taxonomic identities using Kraken, and Bracken was used to determine species abundance of the taxonomic identities assigned by Kraken.37 (link),38 The virulence genes were identified using the ARIBA VirulenceFinder database.39 (link) Isolate genomes were deposited in the European Nucleotide Archive (ENA) under project ID PRJEB8667 (https://www.ebi.ac.uk/ena/browser/view/PRJEB8667 ).
Nebnext ultra 2 fs dna library kit
Manufactured by Illumina
Sourced in United States, United Kingdom
The NEBNext Ultra II FS DNA library kit is a reagent kit used for preparing DNA samples for next-generation sequencing. The kit provides a streamlined workflow for converting DNA into sequencing-ready libraries. It includes reagents and protocols for fragmentation, end-repair, dA-tailing, and adaptor ligation.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq x10, by Illumina (3 mentions)
Wizard dna extraction kit, by Promega (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (3 mentions)
Accublue broad range assay, by Biotium (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (2 mentions)
Dsdna broad range fluorometric quantification assay, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 q5 master mix, by Illumina (2 mentions)
Multiqc, by Babraham Bioinformatics (1 mentions)
Wizard genomic extraction kit, by Promega (1 mentions)
Fastqc, by Babraham Bioinformatics (1 mentions)
Lightcycler 480, by Illumina (1 mentions)
Lc220, by Illumina (1 mentions)
Hiseq x10 with 150 bp paired end chemistry, by Illumina (1 mentions)
8 protocols using nebnext ultra 2 fs dna library kit
1
Whole Genome Sequencing of E. coli Isolates
2
Whole Genome Sequencing Protocol
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The isolates were processed for the extraction of genomic DNA using Wizard DNA extraction kit (Promega; Wisconsin, USA) following manufacturer’s instructions. The extracted DNA was quantified on a Qubit fluorometer (Invitrogen; California, USA) using dsDNA Broad Range quantification assay. Double-stranded DNA libraries 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 on an Illumina HiSeq X10 (Illumina, California, USA).
3
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from single-colony cultures using PureLink™ Genomic DNA Mini Kit (Invitrogen, USA). The DNA integrity was checked by electrophoresis in a 1% (w/v) agarose gel, the DNA purity was estimated using NanoDrop® spectrophotometer (Nanodrop Technologies Inc., USA), and the concentration was determined with a Qubit Fluorometer (Invitrogen, USA).
Subsequently, 1000 ng of DNA was shipped to the Wellcome Sanger Institute (Hinxton, UK) to sequence using Illumina HiSeq-X10 (San Diego, USA). DNA concentrations were confirmed using AccuBlue Broad Range assay (Biotium, Inc., Fremont, USA), followed by normalization and DNA library construction. DNA was sheared into 450–550 bp fragments using Covaris LC 220 ultrasonicator (Brighton, UK), followed by polymerase chain reaction (PCR)-based library preparation using Illumina adaptors and 384-indexed tags (NEBNext Ultra II FS DNA library kit). Afterward, size-selection, amplification, purification, and multiplexing were carried out, and libraries were pooled. Pool was quantified and normalized down to 4 nM using Biomek NXP workstation for (automated liquid handling; California, USA), Agilent Bioanalyzer 2100 (California, USA), and Roche Lightcycler 480) (Utah, USA), before denaturation and loading on the Illumina platform.
Subsequently, 1000 ng of DNA was shipped to the Wellcome Sanger Institute (Hinxton, UK) to sequence using Illumina HiSeq-X10 (San Diego, USA). DNA concentrations were confirmed using AccuBlue Broad Range assay (Biotium, Inc., Fremont, USA), followed by normalization and DNA library construction. DNA was sheared into 450–550 bp fragments using Covaris LC 220 ultrasonicator (Brighton, UK), followed by polymerase chain reaction (PCR)-based library preparation using Illumina adaptors and 384-indexed tags (NEBNext Ultra II FS DNA library kit). Afterward, size-selection, amplification, purification, and multiplexing were carried out, and libraries were pooled. Pool was quantified and normalized down to 4 nM using Biomek NXP workstation for (automated liquid handling; California, USA), Agilent Bioanalyzer 2100 (California, USA), and Roche Lightcycler 480) (Utah, USA), before denaturation and loading on the Illumina platform.
4
Genomic DNA Extraction and Sequencing
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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).
5
Genomic DNA extraction and Illumina sequencing
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Genomic DNA was extracted from single-colony cultures using PureLink™ Genomic DNA Mini Kit (Invitrogen, USA). The DNA integrity was checked by electrophoresis in a 1% (w/v) agarose gel, the DNA purity was estimated using NanoDrop® spectrophotometer (Nanodrop Technologies Inc., USA), and the concentration was determined with a Qubit Fluorometer (Invitrogen, USA). Subsequently, 1000 ng of DNA was shipped to the Wellcome Sanger Institute (Hinxton, UK) to sequence using Illumina HiSeq-X10 (San Diego, USA). DNA concentrations were confirmed using AccuBlue Broad Range assay (Biotium, Inc., Fremont, USA), followed by normalization and DNA library construction. DNA was sheared into 450-550 bp fragments using Covaris LC 220 ultrasonicator (Brighton, UK), followed by PCR-based library preparation using Illumina adaptors and 384-indexed tags (NEBNext Ultra II FS DNA library kit). Afterward, size-selection, amplification, purification, and multiplexing were carried out, and libraries were pooled. Pool was quantified and normalized down to 4nM using Biomek NXP workstation for (automated liquid
6
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.
7
Paired-End Illumina Data Generation
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The generation of paired‐end Illumina data was required to remove errors by polishing the initial noisy ONT‐based genome assemblies. Eight reactions of 500 ng of nuclear genomic DNA each were set up for preparing the short insert Illumina library with the NEBNext Ultra FS II DNA library kit as described by the vendor with the following parameters: the fragmentation, end repair and deoxyadenylation incubation was 3.75 min (fragments ranging from 200 to 1,000 bp). After USER digest, all the reactions were combined and split into five tubes. The library was left size selected with AMPure XP beads at 0.4× ratio followed by another left side selection at 0.2× ratio. The DNA was eluted from both bead fractions (0.4× and 0.4×/0.2×) in 30 µl TE buffer and the concentration estimated by fluorometry. A cycle test was performed with 5 ng of each size‐selected library (0.4× and 0.4×/0.2×) amplified 4, 6, 8, 10 or 12 times with NEBNext Ultra II Q5 Master mix, and the Illumina universal and index primers. Ten cycles produced the optimal amplicon size after a dual size selection (0.77×/0.61×) from the 0.4× size‐selected library fraction (average amplicon size: 473 bp). Four reactions from this library fraction were set up under these conditions, pooled, dual size‐selected, quality checked and sent to our service provider (Custom Science, New Zealand) to be sequenced.
8
Optimized Short-Insert Illumina Library Prep
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Eight reactions of 500 ng of nuclear genomic DNA each were set up for preparing the short insert Illumina library with the NEBNext Ultra FS II DNA library kit as described by the vendor with the following parameters: The fragmentation, end repair and deoxyadenylation incubation was 3.75 min (fragments ranging from 200 to 1000 bp). After USER digest, all the reactions were combined and split into five tubes. The library was left size selected with AMPure XP beads at 0.4X ratio followed by another left side selection at 0.2X ratio. The DNA was eluted from both bead fractions (0.4X and 0.4X/0.2X) in 30 µL TE buffer and the concentration estimated by fluorometry. A cycle test was performed with 5 ng of each size selected libraries (0.4X and 0.4X/0.2X) amplified 4, 6, 8, 10 or 12 times with NEBNext Ultra II Q5 Master mix, the Illumina universal and index primers. Ten cycles produced the optimal amplicon size after a dual size selection (0.77X/0.61X) from the 0.4X size selected library fraction (average amplicon size: 473 bp). Four reactions from this library fraction were set up under these conditions, pooled, dual size selected, quality checked and sent to our service provider (Custom Science, New Zealand) to be sequenced.
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