title: PacBio Raw Data
design: Pure seeds of the fully inbred cowpea accession IT97K-499-35 were sterilized and germinated in the dark in crystallization dishes with filter paper and a solution containing antibacterial (cefotaxime, 50 g/ml) and antifungal (nystatin, 100 units/ml) agents. About 70 g of seedling tissue was collected, frozen in liquid nitrogen, stored at -80C and shipped on dry ice. High molecular weight gDNA was prepared from nuclei isolated from the seedling tissue by Amplicon Express (Pullman, WA). Pacific Biosciences reads were generated at Washington State University (Pullman, WA) following the Procedure and Checklist-20 kb Template Preparation Using BluePippin Size Selection System (P/N 100-286-000-5) protocol provided by Pacific Biosciences and the Pacific Biosciences SMRTbell Template Prep kit 1.0 (P/N 100-259-100). Resulting SMRTbell libraries were size selected using the BluePippin (Sage Biosciences) according the Blue Pippin User Manual and Quick Guide. The cutoff limit was set to 15-50 kb to select SMRTbell library molecules with an average size of 20 kb or larger.  The Pacific Biosciences Binding and Annealing calculator determined the appropriate concentrations for the annealing and binding of the SMRTbell libraries. SMRTbell libraries were annealed and bound to the P6 DNA polymerase for sequencing using the DNA/Polymerase Binding Kit P6 v2.0 (P/N100-372-700). The only deviation from standard protocol was to increase the binding time to 1-3 hours, compared to the suggested 30 minutes. Bound SMRTbell libraries were loaded onto the SMRT cells using the standard MagBead protocol, and the MagBead Buffer Kit v2.0 (P/N 100642-800). The standard MagBead sequencing protocol followed the DNA Sequencing Kit 4.0 v2 (P/N 100-612-400) which is known as P6/C4 chemistry.  Sequencing data were collected in six-hour movies and Stage Start was enabled to capture the longest sub-reads possible.