Qubit high sensitivity dna assay kit

The DNA from the above step was quantified using a Qubit DNA high sensitivity assay kit and Qubit 2.0 machine (Thermo Fisher Scientific, UK). The DNA concentration in each well was normalized to the lowest concentration sample. The DNA was then pooled including negative DNA extraction controls. This library was diluted to 0.4 nM after quantification using the Qubit 2.0, standard curve qPCR and an Agilent high sensitivity DNA kit with the Agilent 2200 Tapestation instrument (Agilent genomics, Santa Clara, US). Library preparation was carried out using dual-indexed forward and reverse primers, with barcodes. Library preparation PCR was performed. The resulting amplicon was cleaned and pooled using AMPure XP beads (Beckman Coulter) as per manufacturer's instructions. Each plate was pooled into an equimolar final library after quantification using a Qubit 2.0 (Life technologies). Library was loaded onto a MiSeq (Illumina) as per manufacturer's protocol for 500 cycle V2 kits with the addition of custom sequencing primers for read 1, read 2 and index 1. Data was analyzed using QIIME software (v1.8.0).

The sample collection is described in Supplementary Materials. RNA was extracted from 200 µl aliquots using stool total RNA purification kit (Norgen Biotek Corp.) following manufacturer’s protocol. RNA quality and quantity were verified following the MIQE guidelines (http://miqe.gene-quantification.info/). The RNA concentration was quantified with the Qubit microRNA assay kit (Invitrogen). The DNA was extracted with the DNeasy PowerSoil Pro Kit (Qiagen) following manufacturer’s instructions with a higher final elution buffer volume (50 µl) to increase DNA concentration which was quantified with Qubit DNA high-sensitivity assay kit (Invitrogen).

RNA was extracted using a stool total RNA purification kit (Norgen Biotek Corp.). The RNA quality and quantity were verified according to the MIQE guidelines (http://miqe.gene-quantification.info/). The RNA concentration was quantified by Qubit with a Qubit microRNA assay kit (Invitrogen). The DNA extraction was performed with a QIAamp DNA stool minikit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. Finally, DNA was eluted in 100 μl of the elution buffer provided with the kit. The DNA quantification was performed with a Qubit DNA high-sensitivity (HS) assay kit (Invitrogen).

The extraction of tumour tissue DNA was performed using the QiAamp mini kit (Qiagen), according to the manufacturer’s instructions. Four to six sections were used for each region. Prior to each EPD, blood was obtained in five 7.5-ml blood tubes containing EDTA. One of these blood tubes was frozen whole. The remaining four were centrifuged at 226 × g for 10 min at 4 °C. Following this, the plasma was collected and centrifuged at 402 × g for 10 min at 4 °C. The whole blood, buffy coat and plasma were then stored at −80 °C until further use. The extraction of germline DNA was performed using 200 µl of buffy coat and was carried out using the QiAamp DNA blood mini kit (Qiagen), according to the manufacturer’s instructions. Qubit DNA High Sensitivity (HS) Assay Kit (Invitrogen) was used to determine quantity DNA. Samples were prepared according to the manufacturer’s instructions and read on the Qubit 4 Fluorometer (Invitrogen).

P. aeruginosa (Pae5 and Pae1505) genomic DNA sequencing libraries were prepared using Nextera DNA library prep kit (Illumina, catalog no. FC-121-1031) and utilizing the Nextera DNA Sample Preparation Index kit (Illumina, catalog no. FC-121-1011) following the manufacturer’s recommended protocol. DNA samples and libraries were quantified using Qubit high-sensitivity DNA assay kit (Thermo Fisher Scientific, catalog no. Q32854). Libraries were pooled in equal quantity and combined to multiplex to make a final library, and quality control (QC) was done again using Qubit quantification kit and Agilent bioanalyzer using high sensitivity DNA chip (Agilent Biotechnology, catalog no. 5067-4626). The final combined library was sequenced using Illumina technology on a NextSeq 500 sequencer using high-output 300-bp single-end read sequencing kit.
Sequencing reads were processed through BBDuk for quality filtering. The filtered reads were analyzed with Juxtaposer (18 (link)) to find mobile elements in the bacterial genomes. Briefly, Juxtaposer searches for recombinant reads relative to the reference genome sequence, which includes reads for the attP and attB products of IGE excision. Finally, attCt software (18 (link)) was used to determine counts for each IGE of attL, attR, attB, and attP, reporting attP/F and attB/F values, where F = attL + attR + 2 attB).

DNA and cDNA quantitation were performed using SYBR Green power mix (ABI) according to manufacturer’s instructions on a CFX384 (Bio-Rad) qPCR machine. Standards for qPCR were generated from conventional PCR purified by MinElute PCR purification (Qiagen). Standards were quantified by Qubit High Sensitivity DNA Assay Kit (Thermofisher Scientific) and diluted with nuclease free water (Ambion) to appropriate concentration for the standard curves. Results from qPCR assays were calibrated using the standard curves.