Similarly, for cohesive end ligated cDNA 11C (Figure ), conditions C5 -C7 displayed significantly lesser NNSA (the least in the case of C7) compared to C8 and C9. The already low end-RFU and NNSA for C5 -C7 digestions in self-annealing ligated cDNA 1C prevented any inference regarding the influence of higher Mg 2+ concentration. However, its beneficial role was noticeable in lower NNSA for C9 conditions over C8 for self-annealing ligation (Figure ). Similarly for cohesive end ligation, digestion conditions involving higher Mg 2+ (e.g., C6 and C7 for Exo III buffer alone or C9 for combined Exo I and III buffer) displayed lesser NNSA than those involving lesser Mg 2+ (C5 and C8) (Figure ). In contrast, the NSA measurements for dual Exo I and III enzymatic digestions involving C5 -C9 conditions for splint-padlock ligated cDNA 4C displayed significantly high end-RFU and NNSA. In some cases (e.g., for C8 and C9 digestions), the NNSA exceeded 100%, implying that the end-RFU from the NSA involving cDNA alone was even higher than that of primer-assisted LRCA. While C5 -C7 conditions for splint-padlock ligations showed lesser NNSA compared to C8 -C9 ones (with least NNSA for C7), the magnitude of both end-RFU as well as NNSA was significantly higher than that of both self-annealing and cohesive end ligation. When compared with dual Exo I and III digestion in ligase buffer (condition C3, Figure ), conditions C5 -C7 demonstrated better (for selfannealing and cohesive end ligation) or comparable (for splint-padlock ligation) mitigation of NNSA.