Tracking the Evolution of the SARS Coronavirus Using High-Throughput, High-Density Resequencing Arrays

Abstract

Mutations in the SARS-Coronavirus (SARS-CoV) can alter its clinical presentation, and the study of its mutation patterns in human populations can facilitate contact tracing. Here, we describe the development and validation of an oligonucleotide resequencing array for interrogating the entire 30-kb SARS-CoV genome in a rapid, cost-effective fashion. Using this platform, we sequenced SARS-CoV genomes from Vero cell culture isolates of 12 patients and directly from four patient tissues. The sequence obtained from the array is highly reproducible, accurate (>99.99% accuracy) and capable of identifying known and novel variants of SARS-CoV. Notably, we applied this technology to a field specimen of probable SARS and rapidly deduced its infectious source. We demonstrate that array-based resequencing-by-hybridization is a fast, reliable, and economical alternative to capillary sequencing for obtaining SARS-CoV genomic sequence on a population scale, making this an ideal platform for the global monitoring of SARS-CoV and other small-genome pathogens.[Supplemental material is available online at www.genome.org and http://www.gis.a-star.edu.sg/homepage/toolssup.jsp.]In April 2003, the SARS coronavirus (SARS-CoV), a singlestranded RNA virus, was identified as the pathogen responsible for Severe Acute Respiratory Syndrome (Drosten et al. 2003;Fouchier et al. 2003). Soon after, the consensus genome sequence for SARS-CoV was published (Marra et al. 2003;Ruan et al. 2003). Our studies have shown that the SARS-CoV mutates at a significant rate. In addition to single nucleotide changes, we observed small deletions of 5-6 nucleotides in some SARS-CoV isolates. Also, we identified several recurrent sequence variants capable of distinguishing genotypes linked to strains originating in different geographical regions (Ruan et al. 2003). As mutations can alter virulence and therapeutic response of viral pathogens, it is important to develop methods for the rapid monitoring of genetic changes in the SARS-CoV in human populations. Moreover, the rapid detection of genetic variants in newly confirmed SARS cases could also provide important clues to the geographic origins of infection, thus facilitating contact tracing.Sequencing of viral genomes has historically used standard dye termination technologies. Recently, sequencing by hybridization to oligonucleotides has been described (Drmanac et al. 1992;Maskos and Southern 1992a;Southern et al. 1992;Schena et al. 1995) and commercialized (Pease et al. 1994;Nuwaysir et al. 2002). These methods have been adapted for studies of genetic diversity in disease, genes, and between species (Drmanac et al. 1998;Hacia et al. 1998;Wang et al. 1998;Hacia 1999;Vahey et al. 1999;Brenner et al. 2000;Drmanac and Drmanac 2001;Fan et al. 2002). Despite these advances, several limitations temper the enthusiasm for sequencing by hybridization, including difficulties in detection of deletions and cost considerations that significantly restrict array modifications and reformatting for optimization (Cutler et al. 2...

Array-Based Resequencing Assay for Mutations Causing Hypertrophic Cardiomyopathy

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