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European Teams Plan to Develop Optical Single-Molecule Sequencing Technology

A recently formed European research consortium working under a new €12 million ($15.5 million) EU grant plans to develop fluorescence-based single-molecule-sequencing methods.
The effort, which began this summer, is one of two projects, or “work packages,” in the area of single-molecule sequencing that are funded under the four-year, multi-project consortium called Revolutionary Approaches and Devices for Nucleic Acid Analysis, or READNA. The consortium is funded through the EU’s 7th framework program for research and technological development.
Announced last week, the consortium involves 10 academic and six corporate research groups in six European countries. The other single-molecule sequencing project relates to nanopore sequencing (see related article, this issue).
Kalim Mir is a group leader at the Wellcome Trust Centre for Human Genetics at the University of Oxford and coordinates the fluorescence-based single-molecule sequencing project. He said that he and his colleagues will focus on two areas: sequencing DNA in real time, and analyzing long DNA molecules in nanochannels with the goal of gaining long-range sequence information.
The consortium did not break down how much funding will go to each project, but Mir said his own laboratory, which is also involved in the nanopore-sequencing effort, receives more than €1 million through READNA.
He said the real-time-sequencing part of the project includes research groups from manifold institutions, including his own, with “a lot of interdisciplinary expertise,” involving labs at the University of Oxford, at Delft University of Technology in the Netherlands, at the University of Southampton in the UK, and at the Technical University of Denmark.
In the first two years of the project the teams plan to explore a number of different sequencing strategies, including ligation-based and polymerase-based sequencing. Mir cited intellectual property-related reasons for declining to reveal details about these strategies. However, he said that “some of the work we will be doing will involve the development of novel labeling and coding systems, and novel types of substrates for DNA sequencing.”
Mir’s group recently published a description of a new sequencing biochemistry it developed that is a hybrid of sequencing by synthesis and sequencing by ligation. He said the researchers plan to do “some further evolution of some of these concepts” as part of READNA.
The article, published online last month in Nucleic Acids Research, includes a proof-of-concept study for the method, called Cyclical Ligation and Cleavage, in which the scientists sequenced three contiguous bases from a template they captured on an ordered microarray.
The method, which involves ligation of labeled DNA/RNA chimeric oligo libraries, identification of the label, and cleavage of the label and undetermined bases, uses standard reagents and arrays and “will therefore be amenable to adaptation and improvement by a community of users for sequencing as much or as little as desired from any genome for which a reference sequence is available,” the authors write in the paper.

“We are trying to do something different, and we will have our own unique position in this area.”

According to the READNA website, another contribution to new sequencing-by-ligation methods will come from Tom Brown, a professor of chemistry at the University of Southampton. His research is focused on “developing new DNA chemistry that can be applied to various biological problems, including novel high-throughput methods of DNA sequencing,” Brown told In Sequence by e-mail. His group has “unique expertise in the synthesis and biophysical characterization of chemically modified oligonucleotides.”
Also participating in the project is Anders Kristensen at the Technical University of Denmark, who will contribute to real-time ligation-based sequencing by synthesis “with a specific role of exploring methods for restricting excitation to surface-attached molecules only,” according to the READNA website.
Other participants include Achillefs Kapanidis’ group at the physics department at Oxford, who will bring to the table their expertise in using single-molecule methods to study transcription. Several assays developed by the group use fluorescence-resonance energy transfer to monitor the kinetics of transcription in real time, and the researchers are able to observe individual steps of RNA polymerase on DNA, he told In Sequence by e-mail.
“Since the ability to see individual steps can be coupled to fluorescence observables that probe the identity of individual bases on the DNA template, it is fairly easy to see that our studies on transcription mechanisms can be extended to provide a valuable contribution to the quest for fast and accurate real-time single-molecule DNA sequencing,” he said.
Also, Cees Dekker’s group at Delft University of Technology will work on “real-time polymerase-based sequencing-by-synthesis motion tracking using Travelling Wave Tracking,” according to the READNA website. Dekker is also working with other researchers on nanopore sequencing under READNA.
According to Mir, READNA’s approaches will differ from those of Pacific Biosciences and of VisiGen Biotechnologies, which was recently acquired by Invitrogen (see In Sequence 10/28/2008). Both companies are also pursuing optical single-molecule real-time sequencing, but READNA’s approach will differ from theirs. “We are trying to do something different, and we will have our own unique position in this area,” he said, without elaborating.
The second focus of the fluorescence-based single-molecule sequencing project will be to develop methods for analyzing single long DNA molecules in nanochannels. “This will enable us, ultimately, to do sequencing on very long DNA molecules and obtain sequence reads in a long-range context,” Mir said. Thus, researchers will be able to obtain information on haplotypes and structural variants such as copy number variations, he added.
In fact, Mir’s lab has already been working on methods for analyzing long DNA molecules by capturing and stretching them on microarrays (see In Sequence 3/27/2007), but the READNA project will “exclusively work with long DNA molecules in nanochannels,” he said.
Expertise for this part of the project will come from Jonas Tegenfeldt at Lund University in Sweden, who is in the process of moving to Gothenburg University, according to Mir. Previously, Tegenfeldt studied DNA nanochannels at Princeton University, where he worked with Han Cao, a founder of BioNanomatrix.
According to the READNA website, Tegenfeldt will use standard epifluorescence techniques “together with more advanced single-molecule detection schemes as well as high-precision localization of bound labels along DNA.”
But the consortium is not the only party working on the analysis of DNA in nanochannels: In the fall of 2007, for example, BioNanomatrix and Complete Genomics began working on ways to integrate DNA sequencing with long-range spatial information, using a grant from the US National Institute of Standards and Technology (see In Sequence 10/2/2007). However, according to Mir, READNA members will be developing a different method for analyzing DNA molecules that are stretched out in channels.
According to Mir, the nanochannels project also brings on board Anders Kristensen and Henrik Flyvbjerg at the Technical University of Denmark, who plan to develop nanofluidic systems and apply algorithms “so we can get higher spatial resolution of the optical signals,” he said.
READNA has been funded since June but details about the consortium were only released by the organizers last week. At this point, the fluorescence-based single-molecule sequencing project does not involve commercial partners. However, Mir said his lab has independently licensed aspects of his own work relating to a sequencing chemistry that differs from the one he published last month to an undisclosed company.

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