Polony Sequence by Ligation Protocol

This protocol is unique compared to the preceding six, in that it is fully automated by the Polonator G.007. Users need only insert the flow cell(s), slide in the kitted reagent chamber, lower the manifold, close the door, and initiate a run.

During the run, a series of anchor primers are flowed through the cells, where they hybridize to the synthetic oligonucleotide sequences at the 3’ or 5’ end of the 17-18 bp proximal or distal genomic DNA tags. Once an anchor primer is hybridized, a mixture of fully degenerate nonanucleotides (‘nonamers’) and T4 DNA ligase is flowed into the cell; each of the nonamer mixture’s four components being labeled with one of four fluorophores, which correspond to the base type at the query position. The fluorophore-tagged nonamers selectively ligate onto the anchor primer, providing a fluorescent signal that identifies the corresponding base on the genomic DNA tag.

Once the probes are ligated, fluorescently labeling the beads, the Polonator images the array in four colors. Each bead on the array will fluoresce in only one of the four images, indicating whether there is an A, C, G, or T at the position being queried. After imaging, the Polonator chemically strips the array of annealed primer-fluorescent probe complex, as well as residual enzyme, using guanidine HCl and sodium hydroxide. This fully automated protocol takes approximately three hours per cycle.

After each cycle of base reads at a given position have been completed, and the primer-fluorescent probe complex has been stripped, the anchor primer is replaced, and a new mixture of fluorescently tagged nonamers is introduced, for which the query position is shifted one base further into the genomic DNA tag. Seven bases are queried in this fashion, with the sequence performed from the 5’ end of the proximal tag, followed by six base reads with a different anchor primer from the 3’ end of the proximal tag, for a total of 13 base pair reads for this tag. This sequence is then repeated for the 5’ and 3’ ends of the distal tag, resulting in another 13 base pair reads. The ultimate result is a read length of 26 bases (thirteen from each of the paired tags), with 4 to 5 bases in the middle of each tag remaining unread. The total length of the run is ~80 hours, with an expected throughput of ~10 Gb per run.