Library preparation and Single Molecule Real-Time (PacBio) sequencing

The SMRT bell was produced using the DNA Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA). The input genomic DNA concentration was measured using a Qubit Fluorometer dsDNA Broad Range assay (Thermo Fisher Scientific, Waltham, MA). 10 μg of gDNA was mechanically sheared to an average size distribution of 15 kb, using a Covaris gTube (Kbiosciences, Hoddesdon, UK). A Bioanalyzer 2100 12K DNA Chip assay (Agilent, Santa Clara, CA) was used to assess the fragment size distribution. 5 μg of sheared gDNA was DNA damage repaired and end-repaired using polishing enzymes. A blunt end ligation reaction followed by exonuclease treatment was performed to create the SMRT bell template. A Blue Pippin device (Sage Science, Beverly, MA) was used to size select the SMRT bell template and enrich for large fragments > 10 kb. The size selected library was quality inspected and quantified on an Agilent Bioanalyzer 12 kb DNA Chip and on a Qubit Fluorimeter (Thermo Fisher Scientific, Waltham, MA), respectively.

A ready to sequence SMRT bell-Polymerase Complex was created using the P6 DNA/Polymerase binding kit 2.0 (Pacific Biosciences, Menlo Park, CA) according to the manufacturer’s instructions.

The Pacific Biosciences RS2 instrument was programmed to load and sequence the sample on 5 SMRT cells (v3.0; Pacific Biosciences, Menlo Park, CA), taking 1 movie of 240 min each per SMRT cell. A MagBead loading (Pacific Bioscience, Menlo Park, CA) method was chosen in order to improve the enrichment of longer fragments.

After the run, a sequencing report was generated for every cell via the SMRT portal, in order to assess the adapter dimer contamination, the sample loading efficiency, the obtained average read-length, and the number of filtered sub-reads.