Abstract
Recent technological advances have greatly increased both the speed and throughput of genome and transcriptome sequencing. The pace of sequencing has further increased with target-enrichment and library barcoding techniques that allow multiplexing of sequencing runs. However, current manual methods for creating libraries do not scale well, limiting the practical investigation of large numbers of samples. To ease the library-creation bottleneck, we describe here a set of protocols, robotics scripts, bulk reagents and instrumentation developed to automate the production of up to 96 DNA fragment sequencing libraries at once. Current protocols for creating sequencing libraries are lengthy, laborious, and not amenable to automation. We describe here new magnetic bead based methods that produce libraries with yield, purity, and size-selection comparable or superior to current column and gel-based protocols. Using these bead-based methods we developed a unique combination of optimized adaptor concentrations and clean-up techniques which increase the yield of libraries from small amounts of DNA by several fold. We also describe robotic scripts for producing 1–96 libraries simultaneously on two commonly used robotic platforms. These protocols accept 10–8000ng of sheared DNA, calculate and dilute barcode adaptors for each library as necessary, automate all intermediate processing steps, and deliver purified libraries ready for amplification off-instrument. An additional script for post-PCR purification of the libraries is also provided. We further describe the Library Builder™ System, comprising a bench top device and kits of plastic tips, tubes and sealed cartridges prefilled with reagents necessary for producing 1 to 13 DNA fragment libraries either with or without size selection. Our analysis of sequencing data shows that libraries produced by all of the above methods are free of excess adaptors which interfere with quantitation, are unbiased, of high complexity, and free from cross-contamination. The protocols herein are provided to the community for use or customization.