Researchers at the Wellcome Trust Sanger Institute and the Babraham Institute announced that they have successfully sequenced single molecules of DNA without the need for library preparation in the December 11, 2012 issue of Biotechniques.

“This is the first time that anyone has been able to directly sequence single molecules of DNA in this way,” commented Paul Coupland, Ph.D., first author from the Wellcome Trust Sanger institute

Developed by Frederick Sanger, Ph.D., and long the “gold standard” of sequencing technologies, Sanger’s original method, published in 1975, led to his receipt of two Nobel Prizes in Chemistry.

In the Sanger method, DNA serves as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.

To date, this and other sequencing methods have required the relatively laborious and time-consuming process of making DNA libraries, or a collection of DNA fragments derived from genomic samples using techniques specific to the sequencing technology being used.

Using the novel sequencing approach described in the current article, the researchers generated sequence data directly from DNA fragments without generating libraries, and using considerably less DNA required using standard methods, going as low as one nanogram of DNA or 500 times less DNA required by some standard practices. The novel technique, the investigators say, has the potential to greatly reduce both DNA consumption and the time it takes to generate sequencing data from small genomes.

The team used the Pacific Biosciences RS (PacBio RS), a system that sequences single molecules of DNA, for the direct sequencing approach. A PacBio RS system SMRTCell, which enables single-molecule, real-time observation of individual fluorophores against a dense background of labeled nucleotide, can yield 35,000–50,000 reads and 100–160 Mb of mapped bases.

The direct sequencing method described by the authors in the current paper generated up to 3000 reads per SMRT cell. While its utility is currently limited to small genomes, the library-less method potentially enables acquisition of sequence data from comparatively low amounts of DNA, even less than 1 ng of input, and within eight hours of sample receipt.

The investigators reported that they obtained sequence data from small circular single-and stranded and double-stranded DNA viral genomes, as well as from linear fragments covering the entire genome of an MRSA strain of Staphylococcus aureus.

The team tried analyzing the genome of one organism using only eight hundred picograms of DNA, over six hundred times less than the quantity used in standard practice. In this example, the PacBio only generated 70 reads, or fragments of sequence, from the genome. Although this is a fraction of the number of reads generated relative to standard library methods, it allowed identification of the specific organism being sequenced and also, the authors say, previously undetectable organisms in metagenomic samples.

“To sequence microorganisms, one needs to be able to grow them in a lab first,” says Tamir Chandra, Ph.D., lead author from the Babraham Institute. “Not only is this time consuming, but sometimes microorganisms do not grow, making it extremely difficult to sequence their genome.

“With this method we can directly sequence these organisms and find out their identity in a short space of time.”

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