Sequencing library preparation and sequencing were performed at the Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, using the Illumina 16S-metagenomics library prep protocol. Paired-end sequencing was performed on a MiSeq using a 600 cycle reagent cartridge. The preparation of the samples for high throughput sequencing involved using a two-step PCR approach according to Illumina protocol. The amplification of the variable region V3 and V4 of bacterial rRNA gene was done using universal bacterial primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing Illumina adaptors as described by Klindworth et al. [22 (link)]. The concentration of the DNA was determined for each sample based on the average of triplicates of concentration (ng/µl) and average library size of 615 bp. Sequencing was done using MiSeq Reagent Kit v3 (600 cycle kit) (Illumina) and the cartridge and reagents were handled according to manufacturer's instructions.
Miseq reagent kit v3 600 cycle kit
Manufactured by Illumina
Sourced in United States
The MiSeq Reagent Kit v3 (600 cycle kit) is a laboratory equipment product designed for use with the MiSeq sequencing system. It provides the necessary reagents and consumables for sequencing runs of up to 600 cycles.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (2 mentions)
16s metagenomics library prep protocol, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Clc genomics workbench software ver 20, by Illumina (1 mentions)
Ez beads, by Promega (1 mentions)
3 protocols using miseq reagent kit v3 600 cycle kit
1
Illumina 16S-Metagenomics Library Preparation and Sequencing
2
Nextera XT Library Preparation Protocol
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Sequencing libraries were constructed using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). Amplification of the tagmented DNA was performed using index primers, and the amplified products were purified with the ACSIA NGSLibPrep Edition (PrimaDiag, Romainville, France), using AMPure XP beads (Beckman Coulter, Brea, CA). The normalization and pooling were performed manually, based on the size of each fragment determined using TapeStation and the DNA concentration was measured with Qubit. Sequencing was performed using an Illumina MiSeq Platform (Illumina, San Diego, CA) with MiSeq Reagent Kit v3, 600 cycle kit (Illumina) according to the manufacturer's instructions.
3
Genomic Analysis of PVL and TSST-1 Strains
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We performed draft whole-genome sequencing of PVL-and TSST-1-positive strains. Genomic DNA was extracted using the bead-beating method with EZ-Beads (Promega K.K., Tokyo, Japan), followed by processing with a combination of magLEAD 6gC and magDEA Dx SV (Precision System Science, Chiba, Japan). DNA library preparation and sequencing were performed using Illumina DNA Prep (Illumina, Inc.., San Diego, CA, USA) and the Illumina MiSeq platform (Illumina, Inc.) for 300 bp paired-end reads using the MiSeq reagent kit v3 600-cycle kit (Illumina, Inc.). Illumina reads were assembled using the CLC Genomics Workbench software ver. 20.0.4 (Qiagen, Hilden, Germany). The assembled contigs were then analysed for multi-locus sequence typing (MLST) using MLST 1.8, SCCmec typing with SCCmec Finder 1.2, and the identification of virulence genes using VirulenceFinder 2.0 (all available on the Centre for Genomic Epidemiology website: http://www. genomi-cepidemiology.org/) [13e15].
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