The long term goal for this core is to provide highly accurate medical sequencing data in a high throughputpipeline at the lowest possible cost to the PPG. The significance of genetic changes identified by this effortwill be evaluated in Project 2 of the PPG, and by investigators from around the world.
Specific Aim : Using high-throughput, directed PCR and DMA sequencing, we will define themutations and polymorphisms present in candidate genes using DNA samples derived from a cohortof 94 AML patients for which we have banked both tumor (bone marrow) and germline (skin)samples. For over two years, a production pipeline for directed PCR and sequencing of selected regions ofindividual human genomes has been in operation at the Genome Sequencing Center (GSC) at WashingtonUniversity. We have developed an automated primer selection system to characterize the optimal tiling pathof amplicons across any region of interest in the genome. The bar-coding of both samples and amplificationprimers preserves integrity as each sample moves through the production pipeline. Any combination ofsample and amplicon is permitted due to the flexibility that we have built into the sample storage systems,automated platforms, and Oracle LIMS databases. Since all amplification primers have universal (forwardand reverse) tails, all sequencing reactions are performed with the same parameters. From 5/06 to 5/07, wehave completed -258,000 reads with a sequencing pass rate of over 90%. We also have developed anautomated alternative chemistry pipeline for amplicons, which are identified during primer selection asdifficult-to-sequence. We analyze the resulting sequence data with two independent, automated systemsthat identify mutations/polymorphisms, insertions and deletions. Rapid manual review of the automatedpipeline analysis reports and sequence data, when necessary, refines and clarifies the automated output.For the PPG effort, we will produce over 15,000 sequencing reads/month. The initial re-sequencing will beperformed on 94 AML tumor samples. When sequence changes that are predicted to alter gene function aredetected in the tumor samples, we will sequence the same amplicons in the matching germline set todetermine whether the change is somatic, or also present in the germline. Once a sequence change isconfirmed to be non-synonymous and somatic, the same amplicon will be sequenced using material from the94 CALGB 'validation' samples to further assess overall mutational frequency. All of these componentstaken together comprise a high-throughput system that produces high-quality medical sequencinginformation at the lowest possible cost.
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