title: PacBio sequence run 3 of pooled PCR amplicons from 14 different R genes in Virola surinamensis DNA: Same sample as 237, but run as a separate library in a different sequencing run
design: Amplicons of 10 selected R genes (10 of the 14 examined in sequence run 1) were produced by first round PCR using Phusion Hot Start II High Fidelity Polymerase and gene specific primers with the 3-prime end attached to the PacBio universal adapter sequences U1 or U2.  A 5 microliter aliquot of PCR product for each gene amplicon was pooled by individual plant sample and cleaned using solid phase reversible immobilization magnetic beads.  These sample amplicon pools (one pool per well of a 96-well plate) were submitted to the Penn State Genomics Core Facility for second round PCR with Kapa HiFi HotStart PCR Kit (KAPA Biosystems) to add a unique PacBio barcode to each sample amplicon pool.  After purification and molarity normalization barcoded amplicons were pooled and processed into SMRTbell Templates.  Circular consensus sequencing (CCS) of these Templates was on a PacBio Sequel System using Sequel chemistry 3.0 for plate two (sequencing run 2).  Movie time was 20 h.  After barcode parsing and CCS generation specifying minimum 10 passes and minimum predicted accuracy of 90% as the criteria for a CCS read, individual amplicons within a sample amplicon pool were deduced by alignment with a reference set of 10 (10 of the 14 examined in sequence run 1) consensus sequences (provided file Amplicons_10_WithoutPrimers.fa) that derived from a prior transcriptome project.