Qiaamp dna mini

DNA extraction from fingernails, peripheral blood lymphocytes, and liver tissue was performed using the DNA extraction DNA Extractor FM Kit (WAKO), Puregene Blood Core Kit (QIAGEN), and QIAamp DNA Mini (QIAGEN), respectively. DNA samples were quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA) and a NanoDrop system (Thermo Fisher, Waltham, MA). Moreover, quality control of each sample was performed using an Agilent 2200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). First, 29-845 ng of genomic DNA was fragmented to an average length of approximately 150 bp using a Covaris S220 sonication device. The fragments were purified, end-repaired, A-tailed, and ligated to adaptors. The libraries were constructed by the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) or NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA). Quality control of the libraries was performed using an Agilent 2200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA).

The total cellular DNA was extracted from patient CD8⁺-depleted PBMCs or resting CD4⁺ T cell ex vivo cultures using the QIA amp DNA Mini or the QIA amp Micro kit (Qiagen), respectively. The total cell-associated HIV-1 DNA was then quantified by ultra-sensitive real-time PCR (Generic HIV DNA cell kit, Biocentric [75 (link)]) according to the manufacturer’s instructions.

We used bisulphite sequencing to determine the level of DNA methylation in the promoter of the PTEN gene. In brief, genomic DNA in the samples was isolated by making use of a QIAamp DNA Mini assay kit (Qiagen) in accordance with the routine assay protocol provided by the kit manufacturer. Then, bisulphite conversion was carried out via adding 5 mol/L of salt sodium bisulphite to each 1.8 µg of the DNA sample. A universal primer free of CpG was made use of for the amplification of both demethylated and methylated promoter of the PTEN gene at 55.0°C annealing temperature. The product of PCR was then quantitatively evaluated through using DHPLC on a WAVE DNA Fragment Analysis System in accordance with the routine assay protocol provided by the manufacturer.

Whole genome sequencing (WGS) was performed on a representative selection of 30 strains based on the MLVA results and epidemiological information.
The DNA of all the 30 Salmonella strains was extracted using a commercial column-based protocol (QIAamp DNA Mini, QIAGEN, Valencia, CA, USA), and purified gDNA was quantified using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Libraries for whole genome sequencing were prepared using a Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA). High-throughput sequencing was performed using a MiSeq Reagent kit v3, resulting in paired-end reads that were 301 bp’s long.
For the analysis of the WGS data, an in-house pipeline was used which included steps for the trimming and quality control check of the reads (Fastp) [34 (link)]. A genome assembly of the paired-end reads was performed using Shovill (https://github.com/tseemann/shovill; accessed on 21 August 2023) with the default parameters.

DNA was isolated from blood with the QIAamp DNA Mini (Qiagen, Milan, Italy) system and stored at −20 °C. IL9 SNPs were selected based on literature review37 (link) and their ability to tag surrounding variants in the HapMap-CEU population of the International HapMap project, NCBI build B36 assembly HapMap phase III ( http://www.hapmap.org). Haplotype-based tagging SNPs were selected by assessing LD blocks from the genes of interest with a pairwise correlation coefficient r² of at least 0.80 and a minor allele frequency higher than 5% in the HapMap-CEU population. Five IL9 SNPs complied with the selection criteria: rs2069885, rs2069882, rs31564, rs1859430 and rs1799962. The applied Biosystems 7500 Fast qPCR system (Life Technologies) was used for SNP genotyping by KASPar assays (KBiosciences, Hertfordshire, UK). Each genotyping set comprised randomly selected replicates of sequenced samples and negative controls. Agreement between original and duplicate samples was ≥99% for all SNPs. Laboratory personnel were blind to the sample status.

DNA isolation from the swab material was performed as previously described using a commercially available DNA extraction kit (QIAamp DNA Mini; Qiagen, Hilden, Germany) (Weissenborn et al., 2010 (link); Wieland et al., 2011 (link)). Real-time human beta-globin-gene polymerase chain reaction (PCR) was performed to demonstrate that samples contained adequate DNA and were free of substances inhibitory to PCR [period-1: LightCycler Control Kit DNA; Roche, Mannheim, Germany; period-2: beta-Globin PCR according to the protocol of Van Duin et al. (2002) (link)]. All samples were analysed by virus-specific quantitative real-time PCR (q-PCR) targeting the VP1 gene for the presence of MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and HPyV10 as previously reported (Weissenborn et al., 2010 (link); Wieland et al., 2014 (link)). The same protocol was applied for the detection of STLPyV using primers 5′-GAAAATCTAGATGCACCTCACAGA-3′ and 5′-TTTGGCACGGATCATATTCA-3′ together with UPL-probe no. 63 (Roche, Mannheim, Germany, Cat no.: 04688627001). Viral loads were expressed as viral DNA-copies per beta-globin gene copy.