DNA-Seq of Tissue Aladar Bladder Tumour (high viral load)
DNA-Seq libraries were constructed at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. Briefly, DNA samples (0.1-1.0 g each) were fragmented in 50 ul microTUBES-50 (AFA fiber screw-cap) on the Covaris S220 sonicator (Covaris Inc.) under the recommended conditions to generate ~300 bp fragments. Libraries were normalized and pooled equimolarly. This step was followed by treatment with the Free Adapter Blocking Reagent protocol (FAB, Cat# 20024145) in order to minimize the presence of adaptor-dimers and index hopping rates.  The library pool was diluted to 2.5 nM and sequenced (one S4 lane, 2x150 cycles) according to Illumina NovaSeq6000 sequencing protocol, using 180 pM loading concentration and 1% PhiX spike-in control. Fragmentation was followed by AMPure clean-up (0.9:1.0 beads:sample ratio). Next, the cleaned-fragmented material (50 ng) was used as input for library construction. Sequencing libraries were constructed using the NEBNext UltraTMII DNA Library Prep Kit for Illumina (Cat# E7645S). We followed the manufacturers protocol with a few modifications. Samples were quantified just before the barcoding step. A set amount of 10-20 ng of sample was used to barcode and generate full adapter-ligated libraries through PCR. PCR was done for only 6-7 cycles of amplification in order to minimize duplicate reads. Barcoding was done using the indexing reagents provided in the NEBNext Unique Dual Index Oligos kit (Cat# E6440S). The final libraries were quantified by fluorescence (QUBIT, ThermoFisher), and sized on the Agilent TapeStation (DNA5000 Screen Tape). A yield of 50-60 ng of library was obtained of an average size ranging from 460-600 bp.