Analysis of whole genomes using mapping by admixture linkage disequilibium (MALD) and candidate genes requires an appropriate set of markers and the ability to accurately genotype hundreds to thousands of patients with hundreds to thousands of markers. Markers appropriate for MALD must have large differences between racial groups. While our first applications of MALD are in African Americans, we expect that other admixed groups like Hispanics will also be explored by ourselves and other groups. To that end, we initially genotyped or obtained data for 742 microsatellite markers in Asians, Hispanics, African Americans and Caucasians. These analyses identified about 300 loci with sufficiently large differences between groups for use in analyses of African American patients and 200 for Hispanics. Determination of approximately hundreds of thousands of microsatellite genotypes for these MALD marker and HIV-1/AIDS projects prompted us to search for more efficient and accurate genotyping strategies. In the first efforts of screening microsatellite markers, samples were handled essentially 96 at a time. Our research and development efforts now allow laboratory assays to be performed in 384-well microtiter plates for high-throughput genotyping. Currently DNA samples are pre-aliquotted into 384-well plates with a pre-polymerase chain reaction (PCR) Hydra pipettor that pipettes all of the samples at once which are then dried down and stored for later analyses. A panel of 11 plates of HIV-1-infected and -exposed individuals has been developed for genotyping replacing the older 96-well format 42 plate """"""""Mega"""""""" panel. The 384-well format """"""""Dense"""""""" panel will likely be used by the most of the PIs in the LGD once they adopt 384-well technology. Genotyping with the Dense panel has been largely automated. A Genetix automated pipettor can add a few microliters of water to each of 384 wells in seconds. Then PCR cocktails for a locus are added with a Packard Multiprobe II robot. Total volumes have been reduced to 7.5 ul and all indications are that we can achieve 3-5 ul ones. These lower volumes are reducing the cost of genotyping reagents such that the increase in going from 96- to 384-well is essentially in increased analysis time, not reagents. Subsequent pooling for multiple analyses is performed with a post-PCR Hydra pipettor, and other genotyping assays (e.g., Fluorescent single base extension and denaturing high performance liquid chromatography) are handled with multichannel pipettors and creative use of the pre-PCR multiprobe II and the post-pcr 384-well hydra. Previously, setting up a few thousand PCR reactions took an hour or two. With these developments in automation and scale reduction it is possible to set up thousands of reactions at the same time. Always on the lookout for bottlenecks the current one is now analyze microsatellite and single base extension products on an automated sequencer. Another bottleneck has been the adoption of the windows NT platform as an industry, making our installed Macintosh base of computers prematurely obsolete and creating an analysis queue. The reduced sample handling has resulted in fewer missing data points that were the result of pipetting errors made in the complex set-up of reactions. Development and implementation of new 1532-well technology which holds tremendous promise will be the future of these efforts, but that equipment and plasticware are in their infancy. The implementation of 384-well matrix-based microtiter plate technology is allowing us to address genetic questions that previously required heroic handling of lots of plates and detailed sample tracking. High Throughput Genotyping of HIV-1-infected and -exposed Individuals
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