EXON RESEQUENCING: The search for sequence variation in human genome
The primary aim of the ExoSeq programme is to obtain a near-complete catalogue of common variation in human genes. In the first instance, we are re-sequencing human exons in 48 Caucasian individuals using high-throughput sequencing of PCR products. For all known genes, novel coding sequences and transcripts, exons and their flanking sequence are extracted from the Vega database which contains manually curated annotation. Primers are designed to encompass the exon and its splice sites and are checked for uniqueness before synthesis. Bi-directional sequencing of amplicons is carried out using Big Dye™ chemistry. Amplicons generated with pre-tested primers typically have a sequencing success rate of 95-99%. We are currently processing 4,000 STSs per week. SNPs are called using an in-house algorithm for detecting heterozygous SNPs, ExoTrace. All high confidence variants detected automatically or passing manual review are deposited in dbSNP monthly. Approximately 46% of SNPs detected are novel, 40% which are rare. Results are made available to the public in GloVar.
Disease gene variation studies have also been initiated in several areas including cardiovascular disease, platelet biology and genes in cognition. Candidate genes are manually annotated and all exons amplified in the relevant patient samples.
We are looking to extend the study to 48 reference individuals from East Africa chosen to represent pre-European ancestry. We are also analysing the variation in other regions of the genome with potential functional importance, including promoters and other regulatory elements. We are exploring variation in regions showing varied levels of conservation between species and in regions that are predicted to be regulatory using computational and experimental methods.