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

Due to the use of tiling probes in resequencing arrays, a single nucleotide mutation at a particular query base could cause a dramatic reduction in the hybridization intensities of neighbouring PM probes up to six bases away (14) . This effect can be measured by studying the NHIP of each query base. We defined the NHIP of each query base as the observed pattern of hybridization intensities of its PM and MM probes and neighbouring (±6 bases from query base) PM and MM probes. To study the effects of sequence variation (mutation) and noise on the NHIP of a query base, we sequenced RNA from H1N1(2009) patient 380 by capillary sequencing and on duplicate microarrays. We compared sequence calls generated using by Nimblescan or by capillary sequencing and compiled a list of true (correct) calls, error calls and 'N' (unknown) calls. In total, of the expected 13 588 bases of the H1N1 virus (based on genome described at http:// www.ncbi.nlm.nih.gov/genomes/taxg.cgi?tax=211044) the microarray called 13 449 bases while capillary sequence was able to call 12 832 bases. Figure 3 shows the NHIPs of a representative set of 40 randomly selected query bases that result in true-nonmutation calls (wild-type calls). We observed that in these NHIPs, the PM probe of the query base together with neighbouring PM probes, have hybridization intensities significantly higher (>1.4-fold) than that of their MM probes in general. We also identified 10 mutations using capillary sequencing in the patient sample. The NHIPs of these 10 true-mutation calls (Figure 4 ) are very different from NHIPs of wild-type calls. The presence of a mutation at the query base created a MM in neighbouring PM probes and caused a drop in their hybridization intensities. The closer this mutation is to the centre of a neighbouring PM probe, the bigger the drop in hybridization intensity. This results in a distinctive dip to the immediate left and right of the centre of the NHIP where the mutation is.