title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template.
abstract: 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.
authors.affiliation: The Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, UK. kalim.mir@well.ox.ac.uk
authors.forename: Kalim U
authors.initials: KU
authors.lastname: Mir
authors.forename: Hong
authors.initials: H
authors.lastname: Qi
authors.forename: Oleg
authors.initials: O
authors.lastname: Salata
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Scozzafava
mesh_terms: D011088:DNA Ligases; D005456:Fluorescent Dyes; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011088:DNA Ligases