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 1: Identification of base queries with ambiguity. On our array platform, the hybridization intensity of each probe is given by the mean and standard deviation of the fluorescence intensities of nine individually scanned pixels. Hence, we define the signal-to-noise ratio (SNR) of a probe as the ratio of the mean to the standard deviation of the intensities of the nine pixels associated with the probe. In our experiments, we found that >95% of all probes had SNR less than T SNR (T SNR = m SNR + 2s SNR , where m SNR and s SNR are the mean and standard deviation of SNR of all probes on the array). The remaining 5% of probes with SNR ! T SNR are unreliable. Hence, base queries with one or more probes with SNR ! T SNR are analysed further in step 2. Furthermore, all base queries whose PM probe in the forward strand and PM probe in the reverse strand are non-complementary, or have weak PM/MM hybridization intensity differentiation (<1.4-fold) are also passed to step 2. Lastly, we also pass all putative mutation calls to step 2 for confirmation.