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

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 addresse...

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Detalles Bibliográficos
Autores principales: Lamble, Sarah, Batty, Elizabeth, Attar, Moustafa, Buck, David, Bowden, Rory, Lunter, Gerton, Crook, Derrick, El-Fahmawi, Bassam, Piazza, Paolo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222894/
https://www.ncbi.nlm.nih.gov/pubmed/24256843
http://dx.doi.org/10.1186/1472-6750-13-104
Descripción
Sumario: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.