Development of microsatellite markers for colony delineation of the invasive Asian subterranean termite in South Florida and Taiwan
For PacBio sequencing, genomic DNA was purified from 15 heads of workers using phenol/chloroform. DNA concentration was firstly pre-checked on NanoDrop (Thermo Fisher Scientific, USA) for purity, with the OD ratios measuring 1.8 and 2.0 for 260/280 and 260/230, respectively. For high-throughput sequencing, the DNA quantity was determined using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). DNA integrity was profiled using the Femto Pulse system and the genomic DNA 165kb Kit (Agilent, USA), showing a distribution at 5-50kb and a major distribution around 21kb. Shotgun library construction was conducted using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, USA). Genomic DNA was then sheared using Megaruptor (Diagenode, USA), and the SMRTbell adaptor-ligated library was subjected to gel size selection at 5-30kb (major at 10kb) using the BluePippin Size-Selection system (Sage Science, USA). SMRT sequencing was performed on Sequel system using Sequel Sequencing Kit 3.0 and SMRT Cell 1M V3 LR kit (Pacific Biosciences, USA) with 20-hour movie runs and 2-hr pre-extension. The polymerase reads were processed with instrument software SMRTlink 9.0 to generate subreads, and also to generate circular consensus reads (CCS) with parameter settings of minimum CCS read length at 10kb, minimum 3 full passes, and minimum predicted accuracy at 99%.