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.

Single-molecule mechanical identification and sequencing

“…Sequencing DNA via successive ligation reactions is currently used in some NGS schemes 26 . We have implemented a similar protocol on our set-up using the detection of the ligation of a complementary oligonucleotide to a growing primer to determine the underlying sequence.…”

Systems biology of embryogenesis

“…Probably the most familiar and frequently used high-throughput technology today is microarrays, which provide large-scale quantitative data for both transcriptional and genomic measurement (Allison et al 2006; Gunderson et al 2005; Schena et al 1995). Among the most rapidly advancing measurement technologies is nucleotide sequencing, with novel ‘Next-Generation’ sequencing methods promising improved efficiency and significantly reduced expense (Eid et al 2009; Mardis 2008; Mir et al 2008; Shendure and Ji 2008). Using reverse transcription techniques these sequencing technologies can also be applied for the de novo interrogation of transcription, small RNA species (Landgraf et al 2007), alternative splicing (Pan et al 2008), and noncoding transcripts (Core et al 2008).…”

Nucleotides and nucleic acids; oligo- and polynucleotides

“…22 A new sequencing method has been described in which the template is captured onto a microarray and then contiguous sequencing carried out using cyclical ligation and cleavage (CycLiC). 23 Mapping of sequence fitness of an aptamer evolved to bind to the protein allophycocyanin has been described which allows for identification of tighter binding sequences. 24 DNA microarrays have also been used for expression profiling, 25 enzyme profiling, using DNA polymerases 26 and to assess copy-number variation in single cells.…”

“…102 These analogues, which the authors described as unlocked nucleic acids (UNA) additively decrease duplex stability and can therefore be positioned strategically to induce a lack of discrimination of a mismatch or to improve hybridisation specificity. The terpyridine analogue of one of these UNA nucleosides (23) has been used to stabilise duplex DNA by incorporation of a (23) into each strand of a duplex such that they are able to stabilise the duplex through complexation with metal ions (see section 3.3). 103 The use of Click chemistry in nucleic acids has become widespread, and is a very convenient way to post-synthetically modify nucleic acids.…”

“…101,298 The copper-mediated hydroxypyridone base pair has also been used as a method for controlling magnetic interactions in duplex DNA by making use of multiple copper base pairs along the DNA duplex. 519 Various metal ions (Ni, Cu, Zn) have been used to mediate terpyridine complexes (23) within duplex DNA to assist duplex stabilisation. 103 Diphenyl-and tetraphenylporphyrin derivatives as Zn(II) complexes have been attached to C5 of a pyrimidine to act as a zipper-structure in duplex DNA, and for energy transfer studies.…”