title: PacBio Ultra-Low Input HiFi whole genome sequencing of Cylas formicarius: adult male
design: High molecular weight (HMW) DNA extraction was performed from a single whole insect (icCylForm1) using the Qiagen MagAttract HMW DNA Kit. DNA shearing was performed on a 10 ng aliquot of DNA using the Diagenode Megaruptor 2 with the 15 kb fragment protocol. After shearing, PCR amplification adapters were ligated onto the DNA, and the DNA was split into two equal concentration aliquots for PCR. The aliquots of DNA were amplified with two PCR master mixes (A and B), and the resulting PCR products from the two amplification reactions were pooled equal mass prior to PacBio library preparation. The sheared, amplified DNA was prepared for PacBio sequencing using the SMRTBell Express Template kit 2.0. The library was size-selected with beads to remove library molecules less than 3 kb in length to generate the final libraries for sequencing. Sequencing was performed on a Sequel II System using Binding Kit v2.0, Sequencing kit v2.0, and SMRT Cell 8M. To target HiFi reads, the library was sequenced using a 30 hour movie time on one SMRTcell. Raw subreads were converted to HiFi data by processing with CCS to call a single high quality consensus sequence for each molecule, using a 99.5% consensus accuracy cutoff.