Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays

Lee, Charlie Wah Heng; Koh, Chee Wee; Chan, Yang Sun; Aw, Pauline Poh Kim; Loh, Kuan Hon; Han, Bing Ling; Thien, Pei Ling; Nai, Geraldine Yi Wen; Hibberd, Martin L.; Wong, Christopher W.; Sung, Wing-Kin

Step 2: Mutation confirmation and base query recovery. A high-confidence mutation call may be a result of coincidental non-specific hybridization of the same MM probe in both strands. As such, it may be inadequate to discern true mutations based solely on differences in the hybridization intensities of PM and MM probes. From our analysis of true-mutation calls made by PBC, we have found that true mutations have a signature NHIP type as per described in Figure 2b . Thus, query bases that result in a mutation call must have this signature NHIP. Finally, to confirm the mutation, we perform nucleotide substitution bias analysis on these query bases. For each of the query bases with NHIP of type described in Figure 2b , we compute the likelihood ' that the observed PM probe (representing the mutation) is indeed the true PM probe of the sample sequence given the hybridization intensity-based ordering of its MM probes (see 'Materials and methods' section). If ' > 2, the query base results in a strong mutation call (represented by upper case base calls 'A', 'C', 'G' or 'T'). If ' > 1, the query base results in a mutation call with weak support (represented by lower case base calls 'a', 'c', 'g' or 't'). Otherwise, they are re-assigned an unknown 'N' call.