We wondered if, and by how much, incorporating NHIP and substitution biases analysis to the PBC results would improve the performance of the PBC algorithm. We observed that more than 70% of the 65 error calls (false mutation calls) made by PBC did not have the characteristic NHIP of a true-mutation shown in Figure 2b . The remaining 30% of the error calls had a NHIP reminiscent of a true-mutation NHIP but did not satisfy the substitution bias rule. Using NHIP and substitution biases analysis together, we were able to reduce the number of false mutation calls to only two. Most of the 4434 'N' calls made by PBC were due to conflicting base calls from the forward and reverse strand. By analysing the NHIP and hybridization intensity reduction order of the query base in the forward and reverse strand individually, we were able to identify the noisy strand and hence, make the base call only from the non-noisy strand. We were able to recover 92% of the 'N' calls made by PBC using this approach.