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

Following PCR product labelling, hybridization and scanning, signal intensities for each probe was generated using Genepix 4.0 software, and annotated using Nimblescan 2.5 software. Initially, the standard Nimblescan software which employs a gain-of-signal approach [PBC algorithm (8) ], was used to determine the viral sequence. The PBC algorithm assumes that the signal intensity of the PM probe (which matches exactly to the sequence in the sample) will be significantly higher than that of the MM probes. While this approach sufficed for $90% of base queries, we observed that the discrimination between the PM and MM signals was not clear for the remaining probes.