Dna hs kit

The cfDNA and genomic DNA (gDNA) were extracted from plasma and lymphocytes, respectively, using a QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) and QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. The DNA concentration was quantified using a Qubit 2.0 Fluorometer with Qubit dsDNA High Sensitivity (HS) Assay Kit (Fisher Scientific, Newark, DE, USA). An Agilent 2100 Bioanalyzer and DNA HS Kit (Agilent Technologies, Santa Clara, CA, USA) were used to assess the fragment length distribution. Only samples containing > 20 ng of cfDNA and >500 ng of gDNA were processed for further analysis. All 81 DNA samples (54 cfDNA and 27 gDNA) were prepared and stored at −20 °C before library construction.

The gDNA from each selected isolate was extracted from overnight cultures in LB medium using DNeasy blood and tissue kit (Qiagen, Ballerup, Denmark) kit, according to manufacturer’s instructions. The quality of DNA was assessed using gel electrophoresis, and the concentration using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Scoresby, Australia). Whole genome sequencing libraries were prepared from separate aliquots of sample gDNA using the Illumina Nextera DNA kit with modifications.
The PCR-mediated adapter addition and library amplification was carried out using customized indexed i5 and i7 adaptor primers (IDT, Coralville, IA, USA), which were developed based on the standard Nextera XT Indexed i5 and i7 adapters (e.g. N701-N729 and S502-S522). Libraries were then pooled and size selected using SPRI-Select magnetic beads (Beckman Coulter, Lane Cove West, Australia). Finally, the pooled library was quality checked and quantified on an Agilent Bioanalyzer 2100 using the DNA HS kit (Agilent, Santa Clara, CA, USA). Whole genome sequencing was performed using an Illumina HiSeq 2500 v4 sequencer in rapid PE150 mode (Illumina, San Diego, CA, USA).
The genome sequences of E. coli has been deposited in NCBI under the bioproject ID PRJNA507325.

Peripheral blood was collected in Streck tubes (Omaha, NE, USA). Plasma was separated within 4 h by centrifuging at 1600× g at 4 °C for 10 min. The supernatant was then centrifuged at 16,000× g at 4 °C for 10 min to remove the remaining cell debris. Oral swab DNA was extracted using prepIT-L2P ORAcollect DNA extraction kit (DNA Genotek Inc., Ottawa, ON, Canada), to be used as the germline control sample. Cell-free DNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), and BM genomic DNA was extracted using a DNA extraction kit (Cwbiotech, China). DNA concentration was estimated using a Qubit fluorometer and a Qubit dsDNA HS analysis kit (Invitrogen, Waltham, MA, USA). cfDNA fragment length was assessed using an Agilent 2100 Bioanalyzer and the DNA HS kit (Agilent Technologies, Santa Clara, CA, USA).

Circulating DNA was isolated from 0.6–1.8 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) and extracted using the DNeasy Blood and Tissue Kit. The DNA concentration was assessed using a Qubit fluorometer (Invitrogen, Carlsbad, CA USA) and the Qubit dsDNA HS (High Sensitivity) Assay Kit. The size distribution of the cell-free DNA (cfDNA) was assessed using an Agilent 2100 Bioanalyzer and the DNA HS kit (Agilent Technologies, Santa Clara, CA, USA).

Liquid biopsy samples (peripheral blood, ascitic effusion, pleural effusion, pericardial effusion and cerebrospinal liquid) were collected in Streck vacutainer tubes (Omaha, NE) and processed within 48 h to separate the supernatant by centrifugation at 1600 g for 10 min. Buffy coat from peripheral blood was kept for DNA extraction as germline control. The supernatant was transferred to microcentrifuge tubes, and further centrifuged at 16,000 g for 10 min to remove remaining cell debris. Separated liquid biopsy samples and buffy coat were stored at −80 °C until DNA extraction. Separated liquid biopsy samples were isolated for cell free DNA (cfDNA) using a QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Buffy coat DNA and FFPE tumor tissue DNA were extracted using the DNeasy Blood & Tissue Kit (Qiagen). DNA concentration was measured using Qubit fluorometer 3.0 (Life Technologies) and the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA, USA). The cfDNA size distribution was evaluated using an Agilent 2100 BioAnalyzer and a DNA HS kit (Agilent Technologies, Santa Clara, CA, USA). The sample quality was assessed based on the following criteria: total amount ≥30 ng for cfDNA samples or ≥100 ng for tumor FFPE samples; fragment length for cfDNA samples was distributed with a dominant peak at 170 bp proximately^{42 (link),43 (link)}.