Improved workflows for high throughput library preparation using the transposome-based Nextera system.

Journal article

BACKGROUND: The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs. RESULTS: We show that an initial bead-based normalisation step can remove the need for quantification and improves sample purity. A 75% cost reduction was achieved with a low-volume modified protocol which was tested over genomes with different GC content to demonstrate its robustness. Finally we developed a custom set of index tags and primers which increase the number of samples that can simultaneously be sequenced on a single lane of an Illumina instrument. CONCLUSIONS: We addressed the bottlenecks of Nextera library construction to produce a modified protocol which harnesses the full power of the Nextera kit and allows the reproducible construction of libraries on a high-throughput scale reducing the associated cost of the kit.

Authors: Lamble, S; Batty, E; Attar, M; Buck, D; Bowden, R

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: BioMed Central
• Journal: BMC biotechnology
• Volume: 13
• Issue: 1
• Pages: 104
• Publication date: 2013-01-01
• DOI: 10.1186/1472-6750-13-104
• EISSN: 1472-6750
• ISSN: 1472-6750
• Language: English
• Keywords: Gene Library; Workflow; Sequence Analysis, DNA; DNA Primers; High-Throughput Nucleotide Sequencing; Clostridium difficile; Automation, Laboratory