Robotic Enrichment Processing of Roche 454 Titanium Emlusion PCR at the DOE Joint Genome Institute

Hamilton, Matthew ; Wilson, Steven ; Bauer, Diane ; Miller, Don ; Duffy-Wei, Kecia ; Hammon, Nancy ; Lucas, Susan ; Pollard, Martin ; Cheng, Jan-Fang

Lawrence Berkeley National Laboratory

关键词: Removal;    Titanium;    Heating;    59;    Processing;   

DOI  :  10.2172/985322
RP-ID  :  LBNL-3658E-Poster
RP-ID  :  DE-AC02-05CH11231
RP-ID  :  985322

美国|英语

来源: UNT Digital Library

【 摘 要 】

Enrichment of emulsion PCR product is the most laborious and pipette-intensive step in the 454 Titanium process, posing the biggest obstacle for production-oriented scale up. The Joint Genome Institute has developed a pair of custom-made robots based on the Microlab Star liquid handling deck manufactured by Hamilton to mediate the complexity and ergonomic demands of the 454 enrichment process. The robot includes a custom built centrifuge, magnetic deck positions, as well as heating and cooling elements. At present processing eight emulsion cup samples in a single 2.5 hour run, these robots are capable of processing up to 24 emulsion cup samples. Sample emulsions are broken using the standard 454 breaking process and transferred from a pair of 50ml conical tubes to a single 2ml tube and loaded on the robot. The robot performs the enrichment protocol and produces beads in 2ml tubes ready for counting. The robot follows the Roche 454 enrichment protocol with slight exceptions to the manner in which it resuspends beads via pipette mixing rather than vortexing and a set number of null bead removal washes. The robotic process is broken down in similar discrete steps: First Melt and Neutralization, Enrichment Primer Annealing, Enrichment Bead Incubation, Null Bead Removal, Second Melt and Neutralization and Sequencing Primer Annealing. Data indicating our improvements in enrichment efficiency and total number of bases per run will also be shown.