As mentioned above, we hypothesized that NSA may originate from inefficient ligation or ineffective exonuclease digestion of precursor unligated substrates and splint oligonucleotides. Therefore, understanding the role of ligation and effective exonuclease digestion was deemed crucial for understanding the origin of NSA in RCA. To study the role of ligation in NSA, three aforementioned ligation approaches, namely self-annealed ligation, splint-padlock ligation, and cohesive end ligation were used in this work to prepare cDNA. Next, the cDNAs prepared from these ligation techniques were subjected to nine different exonuclease digestion conditions listed in the published literature as well as independently conceived in this work. The NSA in RCA utilizing the cDNAs from these ligations and digestion was quantified using highly sensitive quantitative fluorescence-based rolling circle amplification. The possibility of enhancement of NSA in presence of HRCA primer was also investigated. We have also proposed hypothetical models to explain the mechanistic origin of NSA in RCA. Overall, our study aimed at finding conditions that would minimize the absolute (in terms of end-RFU) and relative NSA (with respect to primer-assisted RCA). All experiments were carried out using unmodified (as opposed to chemically phosphorylated) and desalted (instead of HPLC-purified) oligonucleotides to accommodate broad future applications, cost-effectiveness, and to check assay robustness.