Best Practices for Whole Genome Sequencing Using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging particularly due to size, high density of repetitive elements and various levels of ploidy. The Sequel™ System delivers long read lengths which... [ view full abstract ]

Plant and animal whole genome sequencing has proven to be challenging particularly due to size, high density of repetitive elements and various levels of ploidy. The Sequel™ System delivers long read lengths which enable more complete and accurate contiguous assemblies of larger and more complex genomes. With the latest Sequel chemistry v1.2.1, useable bases of 5 – 10 Gb per SMRT© Cell can be achieved with reduced input SMRTbell™ libraries (as low as 5pM). Read lengths averaging 13 kb or greater can be routinely achieved, with the longest reads approaching 50 kb. Furthermore, 50% of useable bases are of reads greater than 15 kb. Here, we present the best practices for achieving long reads for whole genome shotgun sequencing of complex plant and animal genomes. Guidelines for constructing large insert SMRTbell libraries (> 30 kb) to generate optimal read lengths, using the latest chemistry, will be presented. We also describe ways to maximize library yield (per preparation) from 5 µg sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.