Figure 6. Design of the ssODN library and forward strand patterns of the index PCR product.

(a) Design of the ssODN library. Reverse was used in the enrichment and is shown here. The primer highlighted in blue contains a sequence, complementary to the Illumina sequencing primer, which allows skipping one PCR in the NGS preparation (see details in NGS part below). The ssODN library has a staggered design to ensure the diversity while sequencing the constant region. The resulting library consists of 4 ssODNs, which differ from each other in length by+1, +2, and +3 nucleotides (shown in green; see details in NGS part below). There is no substantial homo-dimerization possible between constant regions, which ensures that primarily the variable region will drive conformational changes in the complete aptamer sequences. Additional balancing was introduced to the design of the 5′ constant region to ensure the diversity during NGS (shown in red). (b) Patterns of the forward strands of the index PCR product. The 3′-end was attached to the surface of the flow cell. Arrow: beginning of the sequencing.