PacBio Sequencing Without Library Preparation

Researchers of the Wellcome Trust Sanger Institute have reported DNA sequencing on the PacBio RS sequencer without prior library preparation. As described in an article in BioTechniques last month, the method has so far been applied for sequencing single- and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA modification analysis, as well as linear DNA fragments covering an entire bacterial genome.

The standard library preparation step was skipped and the DNA was directly used in the sequencing reaction. With this approach, the researchers around first author Paul Coupland were able to generate sequencing data with as little as 1 ng of starting material, taking only about 8 hours of time.

“In terms of read length and accuracy, the direct sequencing method is comparable to the standard sequencing protocol on the PacBio”, as Coupland told InSequence. “There are no drawbacks in terms of read length and accuracy because PacBio is already single molecule sequencing, so it’s just skipping the library prep and going straight into the sequencing part.”

Since random hexamers can be applied as sequencing primers, and no growth of organisms is needed during sample preparation, the method can be applied without any a priori information on the organisms in the sample.

Clearly, this technique still needs to be optimised. For example, the sequence yield obtained with this approach is considerably lower than with standard methods (3,000 reads per SMRT cell, in contrast to 35,000 to 50,000 reads for standard methods).

However, the authors think that the technique has great potential for clinical applications, where unknown organisms need to be quickly identified. As Dr. Harold Swerdlow, lead author from the Wellcome Trust Sanger Institute says in their press release: “Our technique can be performed without any prior knowledge of the sequence and with no organism specific reagents, in a short space of time. This makes it a promising alternative for clinical situations such as infection control.”