Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

Massively parallel DNA sequencing methods are rapidly achieving broad adoption by the life sciences research community [1,2]. As the productivity of these platforms continues to grow with hardware and software optimizations, the bottleneck experienced by researchers is increasingly at the front end (the construction of sequencing libraries) and at the back end (data analysis and interpretation) rather than in the sequencing itself.The input material for commonly used platforms, such as the Illumina Genome Analyzer [3], the Roche (454) Genome Sequencer [4], the Life Technologies SOLiD platform [5], as well as for 'real-time' third-generation sequencers such as Pacific Biosciences [6], consists of complex libraries of genome- or transcriptome-derived DNA fragments flanked by platform-specific adaptors. The standard method for constructing such libraries is entirely in vitro and typically includes fragmentation of DNA (mechanical or enzymatic), end-polishing, ligation of adaptor sequences, gel-based size-selection, and PCR amplification (Figure 1a). This core protocol may be preceded by additional steps depending on the specific application, such as cDNA synthesis for RNA-seq libraries [7].Although generally effective, several aspects of the standard method are throughput-limiting or otherwise suboptimal. These include: (1) Labor: there are several labor-intensive enzymatic manipulations with obligate clean-up steps. (2) Time: the protocol requires 6-10 hours from beginning to end, often including an overnight incubation. (3) Automation: although 96-plex, semi-automated processing has been achieved by large-scale genome centers [8], many researchers lack access to the requisite robotic liquid handling systems and/or instruments for parallelized mechanical fragmentation. (4) Sample indexing: incorporation of barcoded adaptors, which enable concurrent analysis of multiple samples and post-sequencing deconvolution, still requires most steps to be carried out on individual