Ammopiptanthus nanus_PacBio_Sequel_20171008
Long-read sequencing was performed at Biomarker Technologies Corporation (Beijing, China) with a PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, CA, USA). The SMRT Bell library was prepared using a DNA Template Prep Kit 1.0 (PacBio p/n 100-259-100) and four 20 kb SMRTbell libraries were constructed. Genomic DNA (10 ?g) was mechanically sheared using a Covaris gTube (Kbiosciences p/n 520079) aiming at DNA fragments of about 20 kb. A Bioanalyzer 2100 12K DNA Chip assay (Agilent p/n 5067-1508) was used to assess the fragment size distribution. Sheared genomic DNA (5?g) was DNA-damage repaired and end-repaired using polishing enzymes. A blunt-end ligation reaction followed by exonuclease treatment was conducted to generate the SMRT Bell template. A Blue Pippin device (Sage Science, Inc., Beverly, MA, USA) was used to size select the SMRT Bell template and enrich large fragments (>10 kbp). The size-selected library was quality inspected and quantified on Agilent Bioanalyzer 12 kb DNA Chip and Qubit Fluorimeter (Life Technologies). A ready-to-sequence SMRT Bell-Polymerase Complex was created using a Binding Kit 2.0 (PacBio p/n 100-862-200), according to the manufacturer¡¯s instructions. The Sequel instrument was programmed to load and sequence the sample on PacBio SMRT cells v3.0 (PacBio p/n 100-171-800), acquiring one movie of 360 min per SMRT cell. The MagBead loading (PacBio p/n 100-125-900) method was employed in order to improve the enrichment of the larger fragments. A total of 13 SMRT cells were processed yielding 64.72G subread sequences.