Nextflex 96 dna barcodes

A PCR of the housekeeping gene GAPDH using the GAPDH-a/GAPDH-b primers was performed in all cases to ensure the integrity of the DNA. We expected a 200 base pairs amplicon size for GAPDH. HPV DNA PCR was performed using the GP5+/6+ primers that target a conserved region of the HPV L1 gene with an expected amplicon size of 150 base pairs. The amplicons, negative controls (containing H₂0), and positive controls (containing a pool of HPV-positive tumors) were visualized by gel electrophoresis. All HPV-positive amplicons were genotyped by sequencing using the KAPA HTP Library Preparation Kit (Kapa Biosystems, Roche Diagnostics, Basel, Switzerland). The NEXTflex-96 DNA Barcodes (Bioo Scientific, Austin, TX, USA) were ligated. DNA quantity was measured by the Qubit dsDNA BR Kit on a Qubit Fluorometer (Invitrogen). The High Sensitivity D5000 kit (Agilent) was used for quality evaluation of the purified library by automated electrophoreses (TapeStation, Agilent). Sequencing was performed on a MiSeq (Illumina, San Diego, CA, USA). The HPV reference genomes (from the papillomavirus database “PaVe” at https://pave.niaid.nih.gov/ (accessed on 1 March 2022)) were used to map the reads.

Pre-verified multiplex PCR primer sets (summarized in Supporting Information Supplemental Table 1 A) were used to amplify the exonic region of TP53 (customized GeneRead DNAseq Mix-n-Match V2 panel, Qiagen GmbH, Hilden, Germany). Target enrichment was processed by means of the GeneRead DNAseq Panel PCR V2 Kit (Qiagen), following the manufacturer's instructions. All purification and size selection steps were performed utilizing Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA). End repair, A-addition and ligation to NEXTflex-96 DNA barcodes (Bioo Scientific, Austin, Texas, USA) was carried out using the GeneRead DNA Library I Core Kit (Qiagen). Amplification of adapter-ligated DNA was conducted using NEXTflex primers (Bioo Scientific) and the HiFi PCR Master Mix (GeneRead DNA I Amp Kit, Qiagen). Next generation sequencing was performed applying 12.5 pM library pools (2% PhiX V3 control) and the MiSeq Reagent v2 chemistry (Illumina, Inc., San Diego, Ca, USA).

Bacterial DNA was purified from FFPE tissue sections using a commercial kit (NucleoSpin Kit, Macherey-Nagel, Düren, Germany) as recently described [9 (link), 10 (link)]. A total of 8100 ng of total DNA was extracted and used for NGS. Purified DNA was fragmented by sonication (M220 Focused-Ultrasonicator; Covaris, Woburn, Massachusetts, USA) and 500 ng of the fragmented DNA was used as input for library preparation with the aid of a SPRI-TE instrument (Beckman Coulter, Krefeld, Germany) with SPRIworks II cartridges and NEXTflex-96 DNA Barcodes (Bioo Scientific, Austin, TX, USA). Library preparation was done without automatic size selection and the resultant libraries were instead manually size selected (peak size 500 bp) with Ampure XP Beads (Beckman Coulter). Finally, the size selected libraries were quantified using the KAPA Library Quantification Kit, Illumina/Universal (KAPA Biosystems, Cape Town, South Africa) and sequenced with an Illumina MiSeq instrument (MiSeq Reagent Kit v2 (500 cycle); Illumina, San Diego, USA). The raw reads were analyzed using RIEMS (zit).
To clarify relevant results of DNA sequences and associated bacterial families, a deliberate mark of all families with a quantity of >40 reads was set and these families were selected (Table 1).