Sequel 2 smrt cell 8 m

HMW-DNA was extracted from ~25 mg snap-frozen tissue, stored at −80 °C until time of extraction, by tissueruptor homogenization according to Circulomics Nanobind Tissue Big DNA Kit Handbook v1.0 (https://www.circulomics.com/store/Nanobind-Tissue-Big-DNA-Kit-p129187130). Long-read libraries were produced using the SMRTbell Express Template Prep Kit 2.0 according to manufacturer’s instructions (Pacific Biosciences, Menlo Park, CA 94025). In brief, 15 µg of genomic DNA per library was sheared into 20 kb fragments using the Megaruptor 3 system. This was followed by removal of single stranded overhangs, DNA damage repair and end-repair/A-tailing before ligation of overhang hair-pin adapters to generate SMRTbell libraries. The libraries were then subjected to nuclease treatment using the SMRTbell Enzyme Clean Up Kit before size fractionation on the Sage Elf system according to the instructions from PacBio. Fractions 2 were selected for sequencing on the PacBio Sequel II instrument using the Sequel II Binding Kit 2.0, Sequel II Sequencing Plate 2.0 and the Sequel® II SMRT® Cell 8 M with 30 h movie time and 2 h pre-extension.

A size-selected DNA sample was sent to the DNA Sequencing Center at Brigham Young University, Provo, Utah, USA. The DNA sample was fragmented with Megaruptor (Diagenode) to 15 kbp and size-selected (>10 kbp) using the Blue Pippin (Sage Science), and prepared for sequencing using the SMRTbell Express Template Preparation Kit 1.0 (PacBio) according to the manufacturer instructions. Sequencing was performed on the Sequel II system (PacBio) using the Sequel II Sequencing Kit 1.0 (PacBio) with the Sequel II SMRT Cell 8M (PacBio) for a 30 h data collection time.

All 23 LRS-unique SVs were validated using long-range PCR followed by sequencing on a PacBio Sequel IIe system. Primers were designed using Primer3Input, and PCR was performed using NEB LongAmp Hot Start Taq 2 × Master Mix. For each PCR product, 500 ng was used as input for the library preparation and the normalized library was prepared according to the manufacturer’s instructions using the SMRTbell barcoded adapter complete prep kit. Finally, the library, with a loading concentration of 80 pm, was sequenced on a PacBio Sequel IIe system using a Sequel II SMRT Cell 8 M (PacBio, Menlo Park, CA, USA) with a movie time of 30 h and 0.7 h pre-extension time.