Methodfully automated process for construction of sequence-ready barcoded libraries for 454

Abstract We present an automated, high throughput library constr uction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method. Background The emergence of next-generation sequencing technolo-gies, such as the Roche/454 Genome Sequencer, the Illu-mina Genome Analyzer, the Applied Biosystems SOLiDsequencer and others, has provided the opportunity forboth large genome centers and individual labs to generateDNA sequence data at an unprecedented scale [1]. How-ever, as sequence output continues to increase dramati-cally, processes to generate sequence-ready libraries lagbehind in scale. The minimum unit of sequence data (forexample, lane or channel) already exceeds the amountrequired for small projects, such as viral or bacterialgenomes, and will continue to increase. As a result, proj-ects with large numbers of samples but small sequenceper sample requirements become increasingly challeng-ing to undertake in a cost-effective manner.The 454 Genome Sequencer uses bead-in-emulsionamplification and a pyrosequencing chemistry to gener-ate DNA sequence reads by synthesis [2]. Longer readsand shorter sequencing run times make the 454 platforma powerful tool for