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

Grading the quality of the sequence calls EvolSTAR employs a two-step process for base-calling (details in 'Materials and methods' section). First, it determines if the gain-of-signal from the PM probe is strong. If strong, then the base sequence is called and annotated as 'high quality'. If the gain-of-signal is ambiguous, or if the base called is different from the expected sequence, then NHIP and nucleotide substitution bias is deployed to verify the sequence call. From empirical experiments and comparison with capillary sequence data, we observed that high quality sequence calls are made when the signal from the PM probe for both forward and reverse strands are at least 40% higher than that of the MM probes.