Nextera dna flex kit

DNA was extracted from whole blood sample using QIAamp^® DNA Mini Kit (Qiagen, Hilden, Germany). A WGS library was prepared using Nextera DNA Flex kit (Illumina, San Diego, CA, USA) according to manufacturer instructions. Paired-end sequencing (150 bp) was performed to mean sequencing coverage of 30× or more. Reads were aligned to the reference genome (GRCh38) and small variants were called using Dragen Bio-IT platform (Illumina, San Diego, CA, USA) and joint-called with GLnexus [12 (link)].
Structural variant (SV) calling was performed with smoove software [13 ]. Annotation was performed using an Ensembl Variant Effect Predictor (VEP) [14 (link)]. All variants were visually inspected in an Integrative Genomics Viewer (IGV) [15 (link)] and breakpoint regions were investigated with PCR and Sanger sequencing. Mobile elements (ME) SVA, LINE1 and Alu were called using MELT software [16 (link)] and annotated with VEP [14 (link)]. Images were prepared using the R programming language. For Figure 1 a trackViewer package was used [17 (link)].

DNA was extracted using a DNA mini extraction kit (Qiagen) following the protocol “Isolation of genomic DNA from Gram-positive bacteria” in “Appendix D: protocols for bacteria,” with the following amendments: the incubation with proteinase K was performed at 56°C for 30 min, and in the final elutions, 50 μl distilled water was added with the full 5 min of incubation. DNA was quantified using a Qubit fluorometer. Sequencing libraries were made using the Illumina Nextera DNA flex kit based on the manufacturer’s instructions (11 ), with several modifications to reduce processing time (see Results). In the first 3 runs, the input DNA to library preparation was normalized to ∼100 ng, but thereafter, we used a range of up to 500 ng DNA. Libraries were sequenced on an Illumina MiniSeq platform with a run time of 13 h (overnight) using the high-output 150-cycle MiniSeq cartridge and the Generate Fastq workflow. Genomes were demultiplexed using the Generate Fastq workflow and the data transferred to an external 1-Tb USB-connected hard drive for further analysis. Ten sequencing runs were performed during this evaluation; the objective of each run is summarized in Table S1 in the supplemental material.

Patients and samples were renumbered with an anonymous study code, after which each patient had a single anonymized MRSA isolate processed for WGS. For each isolate, a 1-μl loopful of growth was inoculated into phosphate-buffered saline to form a suspension and DNA was extracted using the Qiagen DNA mini extraction kit. Sequencing libraries were prepared using the Illumina Nextera DNA flex kit and sequenced with an Illumina MiniSeq system with a run time of 13 h, using the high-output 150-cycle MiniSeq cartridge and the Generate Fastq workflow. Each run contained 3 controls (no-template control, positive control [MRSA MPROS0386], and negative control [Escherichia coli NCTC12241]).

Total DNA extractions were performed on the 200 µL of soil water concentrates, using the DNeasy® PowerSoil® kit (Qiagen). The protocol followed the manufacturer's recommendations: cell lysis, followed by DNA fixation and elution. However, the recommended volume of 100 μL for the elution step was reduced to 60 μL to concentrate the DNA. Final DNA concentrations were quantified using the Invitrogen™ Qubit™ 2.0 fluorometer (Thermo Fischer) and ranged from 0.37 to 1.0 ng µL⁻¹. The extracted DNA was then stored at −20°C until further analyses. The viral communities were determined using shotgun metagenomics sequencing. From the total extracted DNA of soil water samples, library preparations were completed using the Nextera DNA Flex kit (Illumina). Paired‐end sequence reads were generated using the Illumina NovaSeq. 600 (2 × 150 bp).

We sequenced genomic DNA by using an Illumina MiSeq (Illumina, https://www.illumina.com) platform. We used a Nextera DNA Flex Kit (Illumina) for the library preparation, using 500 ng of DNA as input. We assessed library molarity and quality on the Qubit and the tape station using a DNA high-sensitivity chip (Agilent Technologies, https://www.agilent.com). We used the MiSeq Reagent Kit v2 (Illumina) to load libraries. Paired-end reads of 150 bp were generated de novo and assembled by using Shovill 1.1.0, a GALAXY webtool (7 (link)), and the contigs were assembled via the CONTIGuator web server, using the B. ceti TE10759–12 genome (BioProject PRJNA224116) as a reference. We annotated consensus sequences by using the PATRIC genome annotation service (RASTtk) (8 (link)).
We deposited the genome sequences in GenBank under BioProject no. PRJNA728965 (accession nos. CP074683 for chromosome 1 and CP074684 for chromosome 2 of BRSO-2021–230 [BioSample SAMN19108195]; CP075601 for chromosome 1 and CP075602 for chromosome 2 of BRSO-2020–213 [BioSample SAMN19107925]).

Whole-genome sequencing was chosen for this study in order to access the VNTR and CRISPR information. For this, genomic DNA of 20 isolates belonging to the private collection in Wageningen (the Netherlands) was extracted with the Wizard Magnetic DNA Purification System for Food (Promega) according to the instruction of manufacturer. Subsequently, libraries were prepared with the Nextera DNA Flex kit (Illumina) and were sequenced using a HiSeq 2000 sequencer (Illumina) with paired 100 bp read length. Additionally, the DNA of 35 strains was extracted with a standard protocol for cultured cells in the NucleoSpin Tissue Kit (Macherey–Nagel). Libraries were prepared with the Nextera XT DNA kit (Illumina) and sequenced on a MiSeq sequencer (Illumina), generating paired 300 bp read lengths. Raw data for all genomes were de novo assembled with SeqMan NGen v.12.1.0 software (dnastar). Three genomes sequenced with HiSeq were sequenced as well with PacBio technology in order to obtain complete genome sequences (accession numbers: LT853880–LT853887) [47 (link)]. Data from the latter sequencing were used in this study.

PCR amplification of the 16S rRNA gene V3-V4 hypervariable region was performed using dual-barcoded universal primers 318F and 806R as previously described (84 (link)). In brief, amplicon pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA), and the size and quantity of the amplicon library were assessed on the LabChip GX system (PerkinElmer, Waltham, MA) and with a library quantification kit for Illumina (Kapa Biosciences, Woburn, MA), respectively. The PhiX control library (v3) (Illumina, San Diego, CA) was combined with the amplicon library. High-throughput sequencing of the amplicons was performed on an Illumina MiSeq platform using the 300-bp paired-end protocol. Sequence libraries were prepared from the extracted DNA using the Nextera DNA Flex kit (Illumina, San Diego, CA) according to the manufacturer’s specifications. Libraries were then pooled in equimolar proportions and sequenced on a single Illumina NovaSeq 6000 S2 flow cell providing an average of 6.5 million pairs of 150-bp reads per library at the Genomic Resource Center at the University of Maryland School of Medicine.