Targeted PacBio-seq for TERT VNTR6-1 region
PCR amplicons for targeted PacBio sequencing of VNTR6-1 were generated using the LA Taq Hot-Start DNA Polymerase kit (Takara) and M13-tagged primers. These primers capture a genomic fragment of 2,241 bp. The optimized 20 l reactions included 4% DMSO, 0.3 l of LA Taq DNA Polymerase, 2.5 l of 10x LA Taq PCR Buffer, 4 l of 2.5 mM dNTPs, 0.5 l of each 10 M primer, and 25 ng of genomic DNA. PCR conditions included denaturation for 1 minute at 94C, 36 cycles of denaturation for 10 seconds at 98C and combined annealing/extension for 3.5 minutes at 68C, followed by final extension for 10 minutes at 72C. All products were quantified with the Quant-iT PicoGreen dsDNA Assay (Invitrogen) and 5% of the products were analyzed with the TapeStation D5000 Kit (Agilent). The second round of PCR was performed with the LA Taq Hot-Start DNA Polymerase kit and the SMRTbell Barcoded Adapter Complete Prep Kit (PacBio) and utilized the M13 tags incorporated by the first PCR to attach unique barcodes to each sample with primers M13F (NGTAAAACGACGGCCAGT) and M13R (NCAGGAAACAGCTATGAC), where N represents the unique barcode. The 25 l PCR reactions included 4% DMSO, 0.4 l of LA Taq DNA Polymerase, 2.5 l of 10x LA Taq PCR Buffer II, 4 l of 2.5 mM dNTPs, 1.0 l of each 3 M barcoded M13 primer, and 25 ng of product from the first PCR. The PCR conditions included denaturation for 1 minute at 94C, 10 cycles of denaturation for 10 seconds at 98C and combined annealing/extension for 3.5 minutes at 68C, followed by final extension for 10 minutes at 72C.The final amplicons from three 96-well PCR plates (288 samples) were pooled, processed with the Sequel II binding kit 3.1 (PacBio), and sequenced on one SMRT Cell on the Sequel II System (PacBio).