Nucleic Acids Research Advance Access originally published online on November 16, 2008
Nucleic Acids Research 2009 37(1):e5; doi:10.1093/nar/gkn906
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Nucleic Acids Research, 2009, Vol. 37, No. 1 e5
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
The Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN, UK
*To whom correspondence should be addressed. Tel: 01865 287652; Fax: 01865 287533; Email: kalim.mir{at}well.ox.ac.uk
Received October 1, 2008. Revised October 25, 2008. Accepted October 28, 2008.
Next generation sequencing methods that can be applied to both the resequencing of whole genomes and to the selective resequencing of specific parts of genomes are needed. We describe (i) a massively scalable biochemistry, Cyclical Ligation and Cleavage (CycLiC) for contiguous base sequencing and (ii) apply it directly to a template captured on a microarray. CycLiC uses four color-coded DNA/RNA chimeric oligonucleotide libraries (OL) to extend a primer, a base at a time, along a template. The cycles comprise the steps: (i) ligation of OLs, (ii) identification of extended base by label detection, and (iii) cleavage to remove label/terminator and undetermined bases. For proof-of-principle, we show that the method conforms to design and that we can read contiguous bases of sequence correctly from a template captured by hybridization from solution to a microarray probe. The method is amenable to massive scale-up, miniaturization and automation. Implementation on a microarray format offers the potential for both selection and sequencing of a large number of genomic regions on a single platform. Because the method uses commonly available reagents it can be developed further by a community of users.
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