Smrtbell express template prep kit

Highly purified genomic DNA from the isolate FDS-564 was extracted from fungal mycelium grown in a liquid Potato Dextrose Broth for 5 days at room temperature, filtered and freeze-dried before following the modified DNA extraction protocol as previously described (Norgen Biotech Comp., Thorold, Canada). The library was prepared using a SMRT bell Express Template Prep Kit (PacBio, Menlo Park, USA). The library pool was sequenced on one SMRT cell using the PacBio Sequel II platform in the SickKids sequencing facility (Toronto, ON, Canada). The reads were assembled into contigs with the Canu assembler v. 2.1.1 [35 (link)]. The quality of genome assemblies was accessed using QUAST v. 5.0.2 [36 (link)]. The completeness of assembly was estimated with BUSCO v.4.0.5 employing the dataset Ascomycota_odb10 [37 (link)]. Repeat sequences were identified with RepeatMasker v. 4.0.9 [38 ] using the library of repeats for fungi obtained from Repbase [39 (link)]. Gene prediction was executed using Augustus v. 3.4.0 [40 (link)]. Augustus was trained with the gene structures from the representative genome of C. mali 03–8 (GCA_000818155) obtained using the MAKER2 pipeline v. 2.31.11 [41 (link)].

High-molecular-weight DNA (no shearing required) was purified with 0.6× AMPure PB beads (Beckman Coulter) and eluted in Elution Buffer (EB) buffer. Quantification was done on a Qubit (Thermo Fisher Scientific) by using the ‘Broad Range Kit’. A sequencing library was prepared by using the SMRTbell express template prep kit (PacBio) according to the ‘Procedure & Checklist - Preparing > 15 kb Libraries Using SMRTBell Template Preparation Kit’ (PacBio). Library fragments in the 15–50-kb range were size-selected on a Blue Pippin (Sage Science) by using a 0.75% dye-free agarose gel cassette according to the ‘0.75% DF Marker S1 high-pass 15–20 kb protocol’. Library quality was checked on a Fragment Analyzer (Agilent) by using the ‘High Sensitivity Large Fragment 50 kb Kit’. Final yield was measured on a Qubit by using the ‘High Sensitivity Kit’. The library was sequenced on a PacBio Sequel I (PacBio) by using the V2.1 Binding kit, Chemistry kit and three SMRT cells. The loading type was ‘diffusion’ with an on-plate concentration of 6 pM. There was no pre-extension, and data were collected as a 10-h movie.

Genomic DNA was extracted using a Magnetic Plant Genomic DNA Kit (Cat. no: 4992407; Tiangen, China). After quality control, a paired-end library (insert size ∼170 bp) was constructed using a TIANSeq Fast DNA Library Kit (Cat. no: 4992261; Tiangen, China) and then was sequenced by an Illumina HiSeq 2000 sequenator (Illumina, USA). Subsequently, a SMRTbell library (∼20 Kb) was constructed using a SMRTbell Express Template Prep Kit (PN: 100-938-900; PacBio, USA) and then was sequenced by a PacBio Sequel sequenator (PacBio, USA).

Purified genomic DNA (gDNA) was used for library construction with the SMRTbell Express Template Prep Kit (Pacific Biosciences, Cat. No. 101-357-000). In brief, gDNA was mechanically sheared to an average size of 20 kb using a Covaris g-TUBE device (Part No. 520079). In total, 5 μg of sheared gDNA was damage-repaired and end-repaired using polishing enzymes. Blunt-end adapter ligation was used to create the SMRTbell template. Adapter dimers and contaminants were removed using the AMPure XP bead purification system (Beckman Coulter, Cat. No. A63882). A BluePippin size selection system (Sage Science, Cat. No. BLU0001) was used to size select the SMRTbell template and enrich for fragments > 15 kb. Sequencing primer v4 was annealed to the SMRTbell template, and a DNA polymerase/template complex was created using the Sequel Binding Kit 2.1 (Pacific Biosciences, Cat. No. 101-365-900). An additional AMPure XP purification step was performed to remove excess primer and polymerase prior to sequencing. The library was sequenced on a Sequel instrument using SMRT Cell 1M v2 (Pacific Biosciences), taking one movie of 10 hours per cell with the Sequel Sequencing Kit 2.0 (Pacific Biosciences).

A SMRT bell library was produced using the SMRTbell Express Template Prep Kit (Pacific Biosciences 101-357-000). 10 μg of gDNA were mechanically sheared to an average size distribution of 15-20 kb, using a Covaris gTube (Covaris p/n 520079). 3 μg of sheared gDNA was DNA damage repaired and end-repaired using polishing enzymes. A ligation reaction was performed to create the SMRT bell template, according to the manufacturer’s instructions. A Blue Pippin device (Sage Science) was used to size select the SMRT bell template and enrich the big fragments beyond 10 kb. The sized selected library was quality inspected and quantified using a Femto Pulse (Agilent) gDNA analysis assay and on a Qubit Fluorometer respectively. A ready to sequence SMRT bell-Polymerase Complex was created using the P6 DNA/Polymerase binding kit 2.0 (Pacific Biosciences p/n 100-236-500) according to the manufacturer instructions. The Pacific Biosciences RSII instrument was programmed to load and sequence the sample on 1 SMRT cell v3.0 (Pacific Biosciences p/n100-171-800), taking 1 movie of 360 minutes. A MagBead loading (PacBio p/n 100-133-600) method was chosen in order to improve the enrichment of the longer fragments.