Shotgun sequencing
Genomic DNA was isolated from 100 ml in vitro culture of B. duncani (15% parasitemia and 5% hematocrit) using DNasy Blood and Tissue kit (Qiagen; Cat. No. 69506), and quality control along with concentration determination was performed using nanodrop and qubit. DNA integrity was evaluated using Blue Pippin pulse gel and the DNA was then used for library preparation using Pacific Biosciences SMRTbell Express template Prep Kit 2.0 (Cat. No. PN: 100-938-900) according to the manufacturers instructions. The Pacific Biosciences Smart Link software was used to determine loading concentration and proper stoichiometric measurements. The gDNA library was then annealed to the Pacific Biosciences V5 primer for 1h at 20C. The annealed library was then bound to polymerase using Pacific Biosciences Polymerase 2.2 for 1- 4h at 30C and was loaded on to the Sequel II Instrument as an adaptive sequencing run. At least one smart cell was sequenced for each genomic DNA library with a movie time of 30h and a pre-extension of 2h.