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

(i) True-non-mutation-The PM probe (of both strands) of the query base must be a high-confidence call [i.e. it has hybridization intensity ! 1.4-fold that of its mismatch (MM) probes]. Neighbourhood PM probes are also high-confidence calls. Let the mean hybridization intensity of the three nearest PM probes to the immediate left of the mutation base (at position À1, À2 and À3), denoted as {À1,À2,À3} , the mean hybridization intensity of the three PM probes to the far left of the mutation base (at position À4, À5 and À6), denoted as {À4,À5,À6} , the mean hybridization intensity of the three nearest PM probes to the immediate right of the mutation base (at position 1, 2 and 3), denoted as {1,2,3} , and the mean hybridization intensity of the three PM probes to the far right of the mutation base (at position 4, 5 and 6), denoted as {4,5,6} . We impose that {À1,À2,À3} % {À4,À5,À6} and {1,2,3} % {4,5,6} (ii) True-mutation-The PM probe (of both strands) of the query base must have hybridization intensity ! 1.4-fold that of its MM probes. To detect the characteristic dip, we check four mean hybridization intensities: the mean hybridization intensity of the three nearest PM probes to the immediate left of the mutation base (at position À1, À2 and À3), denoted as {À1,À2,À3} , the mean hybridization intensity of the three PM probes to the far left of the mutation base (at position À4, À5 and À6), denoted as {À4,À5,À6} , the mean hybridization intensity of the three nearest PM probes to the immediate right of the mutation base (at position 1, 2 and 3), denoted as {1,2,3} , and the mean hybridization intensity of the three PM probes to the far right of the mutation base (at position 4, 5 and 6), denoted as {4,5,6} . If {À1,À2,À3} < {À4,À5,À6} and {1,2,3} < {4,5,6} , we say this is a dip pattern and the query base is likely to be mutated. (iii) Isolated error/'N'-The PM probe (of both strands) of the query base has hybridization intensity < 1.4fold that of its MM probes. Neighbourhood PM probes are high-confidence calls. (iv) Long consecutive errors/'N's-The PM probe (of both strands) of the query base has hybridization intensity < 1.4-fold that of its MM probes. A majority of neighbourhood PM probes are nonhigh-confidence calls. (v) Unknown error/'N'-All other neighbourhood hybridization profile patterns that do not fall under the previous categories.