Abstract: A method of preparing templates for high throughput nucleic acid sequencing, comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times, i.e. multiple loadings or rounds of template hybridization to improve occupation rates, e.g., of nanowells on a flow cell.

Abstract: The present invention relates to improvements in methods of high throughput nucleic acid sequencing, and in particular to improvements to methods of carrying out extension reactions during pairwise sequencing. The present invention relates to a method for carrying out a strand resynthesis extension reaction during pairwise sequencing, wherein said strand resynthesis extension reaction is carried out between a first sequencing read and a second sequencing read, and wherein said strand resynthesis extension reaction extends one or more immobilised primers to copy a first template strand to generate a second immobilised template strand; characterised in that the strand resynthesis extension reaction is carried out using a non-thermostable strand displacement polymerase at a temperature of less than 55° C., preferably at 38° C.

Abstract: A method of preparing templates for high throughput nucleic acid sequencing, comprising: a) providing a solid support, wherein said solid support comprises a plurality of nucleic acid primers immobilised adaptor nucleic acid sequences which hybridise to one or more of the nucleic acid primers, and the library preparation having a nucleic acid concentration of 400 pM or less; c) allowing single stranded fragments of template nucleic acid to bind to said nucleic acid primers, thereby immobilising said single stranded fragments on said solid support; and d) repeating steps b) and c) at least three further times, i.e. multiple loadings or rounds of template hybridization to improve occupation rates, e.g., of nanowells on a flow cell.