PacBio HiFi and CLR

A HiFi and CLR library was prepared from 13 μg and 10 μg gDNA respectively. Each sample was manually sheared with the Megaruptor 3 instrument (Diagenode, P/N B06010003) with the parameters appropriate for each library size according to the Megaruptor 3 operations manual. Each sample underwent AMPure® PB bead (PacBio®, P/N 100-265-900) purification and concentration before undergoing library preparation using the SMRTbell® Express Template Prep Kit 2.0 (PacBio®, P/N 100-983-900). The HiFi library was prepared according to the HiFi protocol version 03 (PacBio®, P/N 101-853-100) and the final library was size-fractionated to approximately 18 kb using the SageELF® system (Sage Science®, P/N ELF0001), 0.75% cassette (Sage Science®, P/N ELD7510). The CLR library was constructed according to the instructions in the CLR protocol version 01 (PacBio®, P/N 101-693-800), and size selected to > 30 kb using the BluePippin® system (Sage Science®, P/N BLU0001), 0.75% Agarose cassette high pass program and U1 marker (Sage Science®, P/N PAC30KB). HiFi and CLR libraries were quantified by fluorescence (Invitrogen Qubit™ 3.0, P/N {"type":"entrez-protein","attrs":{"text":"Q33216","term_id":"75101668","term_text":"Q33216"}}Q33216) and the size of fractions and libraries was estimated from a smear analysis performed on the FEMTO Pulse® System (Agilent, P/N M5330AA).

The loading calculations for sequencing were completed using the PacBio® SMRT®Link Binding Calculator v8.0.0.78867. Sequencing primers v2 and v4 were annealed to the adapter sequence of the HiFi and CLR libraries respectively. The libraries were bound to the sequencing polymerase with the Sequel® II Binding Kit v2.0 (PacBio®, P/N 101-842-900). Calculations for primer and polymerase binding ratios were kept at default values for the respective library types. Sequel® II DNA internal control was spiked into each library at the standard concentration prior to sequencing. The sequencing chemistry used was Sequel® II Sequencing Plate 2.0 (PacBio®, P/N 101-820-200) and the Instrument Control Software v8.0.0.78867.

Each library was sequenced on one Sequel II SMRT®cell 8 M. The run parameters for sequencing the HiFi library were the following: diffusion loading, 30-hour movie, 2-hour immobilisation time, 4-hour pre-extension time, 35 pM on plate loading concentration. The run parameters for sequencing for the CLR library: diffusion loading, 15-hour movie, 2-hour immobilisation time, no pre-extension time, 30pM on plate loading concentration. HiFi reads were generated from the output of the HiFi run using SMRT Tools v 8.0.0.80529.