Nextera xt dna sample preparation kit

To characterize microbial communities and subsequently identify antibiotic resistance elements in samples, libraries were prepared using Nextera XT DNA Sample Preparation kit (Illumina, Inc., San Diego, CA, United States). Sequencing libraries were generated using 1 ng of DNA, according to the manufacturer’s instructions, with a gel size selection modification using Coastal Genomics’ Ranger Technology (Uyaguari-Diaz et al., 2015 (link)). Metagenomic sequencing was performed on a MiSeq platform (Illumina, Inc., San Diego, CA, United States) using an Illumina MiSeq V2 2 × 250 bp paired-end reagent kit. Seventeen water samples were multiplexed on each MiSeq cartridge. To balance the diversity and account for batch effect in each sequence run, we included five samples from agricultural, five samples from urban, five samples from protected watersheds, one negative or background control, and one mock community (Peabody et al., 2015 (link)). Genomic DNA from known bacterial species were pooled in equal molar amounts and processed like indicated in the Nextera XT DNA Sample Preparation kit (Illumina, Inc., San Diego, CA, United States). Raw bacterial reads were created and are available as part of a large-scale Watershed Metagenomics project, BioProject ID: 287840¹.

WGS was performed in each institution as follows: (i) ISCIII, DNA was extracted using the QIAamp DNA minikit (Qiagen, Hilden, Germany), genomic DNA paired-end libraries were generated using the Nextera XT DNA sample preparation kit (Illumina, Inc., San Diego, CA), and the libraries were sequenced using the Illumina NextSeq 500 sequencer system with 150-base paired-end reads (Illumina) and (ii) RIVM, DNA isolation and sequencing using the Illumina HiSeq 2500 was performed by using BaseClear B. V. (Leiden, the Netherlands), libraries were generated using a Nextera XT DNA sample preparation kit (Illumina), and sequencing yielded 150-base paired-end reads.
Short reads were quality trimmed and de novo assembled using Qiagen CLC Genomics Workbench software v7.0.4 (CLC Bio, Aarhus, Denmark), with the default parameters and a minimum contig length of 1,000 bp. Quality assessment of genome assemblies was done by using QUAST v5.0.2 (67 (link)). A quality assembly report is included as supplementary material (see Data Set S1 at https://doi.org/10.6084/m9.figshare.22794125.v3).

Total RNA was amplified using the Ovation RNA-Seq System v2 assay (Tecan Genomics) according to the manufacturer’s protocol. The libraries were prepared with the Nextera XT DNA Sample Preparation kit (Illumina Technologies). In total, 1 ng of cDNA was resuspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Amplicon Tagment Mix), incubating at 55°C for 10 minutes. Neutralize Tagment (NT) buffer was then added to neutralize the samples (part of the Nextera XT DNA Sample Preparation kit, Illumina Technologies). Libraries were prepared by PCR with the random primer Nextera PCR Master Mix and 2 Nextera Indexes (N7XX and N5XX) according to the following program: 1 cycle of 72°C for 3 minutes; 1 cycle of 98°C for 30 seconds; 12 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 1 cycle of 72°C for 5 minutes. The size of the libraries for each sample was measured using the Agilent HS DNA chip (Agilent Technologies). The samples were placed in a pool. The concentration of the pool was optimized to acquire at least 25 million to 30 million reads per sample. Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer protocol using Paired-End Reads with a read length of 75 bps. RNA-Seq analysis was performed by BioWardrobe, as previously described (56 (link)).