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

BMC Biotechnology

Methodology Article

Bassam El-Fahmawi¹  Derrick Crook²  Moustafa Attar³  Elizabeth Batty³  Rory Bowden³  Gerton Lunter³  David Buck³  Paolo Piazza³  Sarah Lamble³ 

[1] Axygen Inc., A corning Subsidiary, 33120 Central Avenue, 94587, Union City, CA, USA;Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, OX3 9DU, Oxford, UK;Oxford NIHR Biomedical Research Centre, John Radcliffe Hospital, Headley Way, OX3 9DU, Oxford, UK;Wellcome Trust Centre for Human Genetics, OX3 7BN, Oxford, UK;

关键词: Nextera;    High-throughput;    Library preparation;    Sequencing;    Normalisation;   

DOI  :  10.1186/1472-6750-13-104

 received in 2013-03-26, accepted in 2013-10-25,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.

【 授权许可】

CC BY   
© Lamble et al.; licensee BioMed Central Ltd. 2013