Best Practices for Whole Genome de novo Sequencing with Long-Read SMRT Sequencing

With the introduction of our latest chemistry release (P6-C4), PacBio has made significant strides with single-molecule sequencing whereby read lengths averaging between 10 and 15 kb can be achieved with extreme reads in the distribution of > 40 kb. Furthermore, coverage requirements to achieve 99.999% accuracy (QV50) has been reduced (30x) coupled with an increase throughput from the PacBio^® RS II platform (500 Mb – 1 Gb per SMRT^®Cell) making larger genome projects more tractable. These combined advancements in technology deliver results that rival the quality of Sanger “clone-by-clone” sequencing efforts; resulting in closed microbial genomes and highly contiguous de novo assembly of complex eukaryotes on multi-Gbase scale using SMRT Sequencing as the standalone technology.

We present here the guidelines and best practices to achieve optimal results when employing PacBio-only whole genome shotgun strategies for plant and animal genomes with specific examples. We further describe SMRTbell™ library preparation and purification methods to obtain suitably long insert libraries to obtain optimal sequencing results. The benefits of long reads are demonstrated by the highly contiguous assemblies yielding contig N50s of over 5 Mb compared to similar assemblies using next generation short-read approaches. Finally, guidelines will be presented for planning out projects for the de novo assembly of large genomes.