While contemplating the origin of the NSA in these substrates, it was immediately apparent that the relatively lower end-RFU and NNSA in self-annealing ligation were possible due to its utilization of only one precursor oligonucleotide (compared to two for the splint-padlock and cohesive end ligations). As a result, the possibilities of intermolecular interactions between cDNA and precursor oligonucleotides were lower compared to the other ligations discussed in this work. As a result, selfannealing ligation has the lowest possibilities of intermolecular interactions that could lead to NSA among the three substrates 1C, 4C, and 11C, causing overall lower end-RFU and NNSA in it. The possible origin of NSA in splint-padlock ligation in all the conditions might be because of the high thermodynamic stability between splint and padlock (or cDNA) oligonucleotides. As observed in the denaturing PAGE, the splint could not be completely denatured or removed from the cDNA 4C by using heat (during snap cooling) or enzyme digestion (as indicated by high NSA magnitude), causing it to function as a primer and leading to NSA during the RCA. In the case of cohesive end ligation, the formation of a strand displacement intermolecular complex between unligated precursors and circular DNA using the cohesive ends could be the reason for NSA and discussed in detail later in this study.