Greater than 10 kb Read Lengths Routine when Sequencing with Pacific Biosciences' XL Release

Abstract

PacBio's SMRT® Sequencing produces the longest read lengths of any sequencing technology currently available. There have been a number of recent improvements to further extend the length of PacBio® RS reads. With an exponential read length distribution, there are many reads greater than 10 kb, and some reads at or beyond 20 kb. These improvements include library prep methods for generating >10 kb libraries, a new XL polymerase, magnetic bead loading, stage start, new XL sequencing kits, and increasing data collection time to 120 minutes per SMRT Cell. Each of these features will be described, with data illustrating the associated gains in performance. With these developments, we are able to obtain greatly improved and, in some cases, completed assemblies for genomes that have been considered impossible to assemble in the past, because they include repeats or low complexity regions spanning many kilobases. Long read lengths are valuable in other areas as well. In a single read, we can obtain sequence covering an entire viral segment, read through multi-kilobase amplicons with expanded repeats, and identify splice variants in long, full-length cDNA sequences. Examples of these applications will be shown.