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

From a broader perspective, this study has highlighted the feasibility of using resequencing microarrays for high-throughput full genome sequencing of viruses. In our application, resequencing microarrays are relatively low-cost, costing only a 10th that of a 454 run, and equivalent to that of a traditional capillary sequencing run. However, through multiplexing, our system can generate full genomes of 24 different H1N1(2009) samples in 30 h. In comparison, capillary sequencing and next-generation technologies such as 454 may obtain full genomes of only one or two different samples in the same time-frame. In practice, capillary sequencing is labour-intensive and thus impractical for large-scale full genome sequencing of viruses in an outbreak. In our experience with the 454 system, much of the amplified material is still human (as the bulk of the patient sample material is human RNA with very little influenza RNA), requiring very deep sequencing to obtain a complete flu genome sequence, with one compartment of a run not yielding sufficient viral information. Furthermore, assembly of the sequence fragments is required before any analysis can be done. Any abnormalities or gaps in the assembly would then require additional runs of 454, incurring more cost and time. Hence, our approach based on resequencing microarrays presents a cost-effective and efficient solution for high-throughput full genome sequencing of viruses.