Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation

Abstract

We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400-500 ng of sheared DNA for the library preparation.

Figure 1. The coverage plot of M13mp18 viral genome after sequencing wih a single SMRT cell

(Left) M13mp18 ssDNA sequenced using the M13 forward sequencing primer. (Middle) M13mp18 dsDNA sequenced using the M13 forward and reverse sequencing primers. (Right) Coverage of M13mp18 dsDNA sequenced using random hexamer primers.

Figure 2. SMRT View genome browser, showing sequence data mapped against M13mp18

A single >10 kb read, longer than the entire M13mp18 circular genome, is highlighted.

Figure 3. SMRT View genome browser, showing kinetic information elucidating modified bases

Four instances of m6A methylation in GATC motifs as detected with the PacBio base-modification analysis software (Motif Finder).