Dna high sensitivity kit

DNA was extracted from blood using the QIAcube system with QIAmp DNA kit (Qiagen, Hilden, Germany), and 10 ng were used for sequencing by NGS technology. The customized designed SNP panel was composed of n =139 SNPs (Supplementary Table S1), present in n =89 amplicons (size range 125–375 bp). This panel permitted to identify 417 genetic variants in total (Supplementary Table S1). The analysed SNPs were selected based on an extensive review of articles on the association between psoriasis and SNPs or response to biologics [7 (link),9 (link),12 (link),18 (link),22 (link),23 (link),24 (link)].
NGS was performed using the Ion GeneStudio™ S5 Plus platform (Thermo Fisher Scientific, Massachusetts, USA). Libraries were amplified by the Ion AmpliSeq™ Library kit Plus (Thermo Fisher Scientific) and quantified using the Qubit 4 Fluorometer and 2100 Bioanalyzer with dsDNA HS assay and High Sensitivity DNA kit (Thermo Fisher Scientific), respectively. Sequencing data were processed with the Ion Torrent Suite software v.5.10.
Positive calls were selected with a read depth >30X and allelic frequency higher than 0.3 (range 0–1.0). Reads were aligned to human genome sequence (build GRCh37/human genome 19). Variants were collected using Variant Caller plugin and systematically evaluated, filtered, and annotated using tailored R scripts. Variants’ annotations were finally verified using ANNOVAR.

A pathologist evaluated histological and pathological features of the cases included in both cohorts and the tumor area was identified on the hematoxylin and eosin (H&E) stained slide before proceeding to the experimental procedure for both stdFFPE and coldFFPE samples. Data were analyzed anonymously. Five 8-μm thick sections were dissected and DNA samples were purified from all the 38 FFPE pairs for a total of 72 DNA samples. In addition, in 14 cases from cohort B for which at least 6 months elapsed from collection paired stdFFPE and coldFFPE samples underwent a second DNA extraction 6 months after collection/fixation.
DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, following an overnight 56°C tissues lyses allowing a complete tissue digestion without fragmenting the DNA. DNA was eluted in 40 μL of nuclease-free water and quantified using the Qubit 3.0 Fluorimeter (Life Technologies, Wilmington, DE, USA) following the protocol of High Sensitivity DNA Kit (Life Technologies, Eugene, OR, USA). To check for any possible contaminant on the purified samples, DNA concentration was also evaluated using the DeNovix DS-11 UV-Vis Spectrophotometer (DeNovix, Wilmington, DL, USA) to obtain the 260/230 and the 260/280 nm ratios.

Whole DNA was extracted from 0.25 g of each fecal sample using the QIAamp PowerFecal DNA kit (Qiagen Group, Hilden, Germany), following manufacturer’s instructions, quantified on a Qubit (Life Technologies) using the High Sensitivity DNA kit, and amplified by the DNA Services Laboratory, Roy J. Carver Biotechnology Center, University of Illinois, Urbana-Champaign, IL, USA. Library preparation for Illumina MiSeq sequencing of the V1-V3 regions of the 16S rRNA gene was carried out using the primer pair 28F and 519R for the PCR amplification of bacteria, while the 5′-GCATCGATGAAGAACGCAGC (forward) and 5′-TCCTCCGCTTATTGATATGC (reverse) primers were used to amplify the ITS1-ITS2 region of fungi (protocols found in references 121 (link) and 122 (link); further information in Text S1 in the supplemental material). Sterile water and human fecal DNA samples were also included as negative and positive controls during each step.

Molecular analyses were performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections using three representative 8-μm-thick sections of tissue samples. The pathologist selected an area with more than 50% of dysplastic cells. DNA was extracted after manual dissection using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and quantified using the Qubit 2.0 Fluorometer (Life Technologies/Thermo Fisher Scientific, Wilmington, DE, USA) following the protocol of High Sensitivity DNA Kit (Life Technologies, Eugene, OR, USA).
All the 102 adenomas were checked for hotspot mutations in KRAS, BRAF, NRAS and PIK3CA genes using the mass spectrometry matrix-assisted laser desorption ionization time of flight method with the MassARRAY System (Agena Bioscience, Hamburg, Germany) with Myriapod Colon Status (Diatech Pharmacogenetics, Jesi, Italy) (Additional file 1: Table S1).

Genomic DNA from tumors and blood was purified using the QIAGEN Puregene Kit (catalog 158445). DNA from buffy coats was purified using QIAGEN DNeasy Blood & Tissue Kit (catalog 69504). The Genomic DNA was sheared with Ion Shear Plus Reagents (Ion Plus Fragment Library Kit) and size-selected with Agencourt AMPure XP beads (Beckman Coulter). DNA fragments with a base pair peak of 100–150 bp were ligated with Ion adapters, purified with Agencourt AMPure XP beads, and PCR-amplified. Then, 750 ng of the adapter-ligated DNA library was hybridized to SureSelect capture library (Agilent SureSelect XT Mouse All Exon Kit) for 20 hours at 65°C. The hybrid capture library was selected using Dynabeads MyOne Streptavidin T1 beads (Life Technologies). The captured library was amplified and purified with AMPureXP beads, and quality was assessed on the High Sensitivity DNA Kit (Life Technologies) on the Agilent Bioanalyzer. We selected a 220 bp peak using E-Gel SizeSelect 2% agarose gel (Life Technologies). The final library was purified and quality assessed on High Sensitivity DNA Bioanalyzer chip (Life Technologies). Templates were prepared using the Ion PI Hi-Q Chef Kit (Life Technologies) on the Ion Chef platform and sequenced on an Ion Proton Sequencer on a PI v3 chip using Ion PI Hi-Q Sequencing 200 Kit (Life Technologies).

Total DNA was quantified on Qubit 3.0 Fluorometer (Invitrogen, USA) using a DNA high sensitivity kit (Invitrogen, USA) and NanoDrop^® ND-1000 ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, USA). The quality of the DNA was also checked on 0.8% agarose gel.