Complex Mixtures of Targeting Oligonucleotides Unlock the Power of Next-Generation DNA Sequencers

A group of researchers at Harvard Medical School, Virginia Commonwealth University, Agilent Technologies Inc. (NYSE: A), Codon Devices Inc., and Stanford University has used complex mixtures of oligonucleotides in the form of oligonucleotide library synthesis to overcome a major bottleneck in realizing the potential of the newest generation of DNA sequencers to reduce costs and increase throughput on a grand scale.

Their findings are reported today in the online version of the journal Nature Methods. The article, Multiplex Amplification of Large Sets of Human Exons, can be accessed at www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth1110.html.

The full potential of the new DNA sequencers is severely constrained by the lack of massively parallel “front end” methods to replace traditional polymerase chain reactions, according to the article. The team synthesized complex oligonucleotide probe mixtures using programmable “inkjet” technology, then applied the oligonucleotide probe mixtures to simultaneously capture and amplify approximately 10,000 human exons in a single reaction. The researchers then demonstrated how this technique can be integrated with ultra-high-throughput sequencing for economical, high-quality targeted variation discovery.

“Agilent adapted its in-situ oligonucleotide synthesis technology to oligo library synthesis, and the oligo length and low error rates had already been helping us with massively parallel synthetic constructs,” said co-author George Church, Ph.D., professor of genetics, Department of Genetics, Harvard Medical School. “It was clear that this approach could also be harnessed for second-generation sequencing primer tools.”

“There are many creative ways to use complex oligo libraries, said co-author Jay Shendure, M.D., Ph.D., assistant professor, Department of Genome Sciences, University of Washington (previously at Department of Genetics, Harvard Medical School). ”The high accuracy and length of these probes really open up some new possibilities"

The article compares the new oligo library protocol to conventional targeted discovery of genetic variation using Sanger sequencing of PCR amplicons. With PCR as the “front end” enabling the amplification of discrete regions that can be covered by an individual Sanger sequencing read -- about one kilobase -- the bottleneck becomes apparent when used with new sequencing techniques.

The article provides an example: “...one gigabase of sequencing capacity would enable the resequencing of 50,000 x 1kb regions at 20x coverage. However, this would still require one to perform 50,000 PCR reactions, as well as to normalize and pool amplicons. Our ability to fully leverage the power of next-generation sequencing technologies is markedly crippled by the lack of a corresponding ’front end’ targeting technology, analogous to PCR, that is matched to a scale at which they will operate.”

“The ability to synthesize a complex mixture of high-quality oligos in a single tube makes a wide range of experiments economically feasible for the first time,” said co-author Emily Leproust, Ph.D., R&D Chemistry manager, Genomics Solutions Unit, Agilent. “Agilent Oligonucleotide Library Synthesis can produce 55,000 oligos in the same tube at a fraction of the cost of individually synthesized oligos. In addition, Agilent’s proprietary chemistry development enabled the synthesis of oligo length of 100 or more base pairs in high quality. A number of researchers are developing exciting new applications using these oligo libraries through an Early Access Program.”

“We anticipate that the successful development of highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole genome sequencing,” the article states.