Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template.
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.
Mir
Kalim U
KU
The Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, UK. kalim.mir@well.ox.ac.uk
Qi
Hong
H
Salata
Oleg
O
Scozzafava
Giuseppe
G
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
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011088:DNA Ligases