E. Nanthakumar, W. Cunha, G. Ederer, C. Manaster, A. Roter, D. Gurevitch, A. Watson, R. Idury, L. Cardon and J. Hall.
Sequana Therapeutics, La Jolla, CA.
We have developed a high-throughput genotyping system combining fluorescent fragment analysis technology, robotics systems, a sophisticated in-house database and informatics tools. This system allows a single technician to generate up to 10,000 high quality genotypes/week. Genomic DNA samples are arranged into bar-coded 96-well plates which contain gel variation, plate orientation and identification controls for downstream QC purposes. PCR reaction master mixes are prepared and plated into DNA plates using a Packard robotic system. PCR products undergo QC by agarose gel analysis and then move to an automated in-house system (Bandit) for unattended post-PCR pooling of up to 20 markers in a single multiplex set. Bandit is composed of a CRS robotic arm, a Sagian 96-well multipette robot and plate hotels. Following acrylamide gel analysis on ABI 373 or 377 automated sequencers two in-house scripts, Mover and Monitor, automatically transfer gel files to file servers and dedicated Genescan (ABI) automatic analysis stations. After confirmation of lane tracking, Genescan files are checked for sizing errors and electropherograms are imported into our database using an in-house Size Standard Validation Tool. Allele assignment is performed using Genotyper software (ABI) and allele size calls are imported into the database. When a marker has been completed across a population the genotype data is analyzed with our Allele Analysis Tool (AAT) for automatic binning and allele assignment (conversion of allele size to letter code). AAT allows data to be displayed by individual 96-well plates to enhance troubleshooting of poorly binning markers. Another in-house tool, Norman, may be used to automatically normalize data in the database using gel variation controls. Normalization also allows the comparison of data run on different instrument platforms such as the ABI 373 or 377. The above operations are run blind to pedigree information. A final QC process consists of analyzing the data with Superchecker, an in-house Mendelian inheritance checker which handles multi-generational families. Mendel errors are resolved with our Finishing Tool which displays pedigree structure, allele statistics and electropherogram data.
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Updated 2/5/2009