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- Title
Improved workflows for high throughput library preparation using the transposome-based nextera system.
- Authors
Lamble, Sarah; Batty, Elizabeth; Attar, Moustafa; Buck, David; Bowden, Rory; Lunter, Gerton; Crook, Derrick; El-Fahmawi, Bassam; Piazza, Paolo
- Abstract
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.
- Subjects
NUCLEOTIDE sequence; GENOMICS; GENOMES; SCIENTIFIC apparatus &; instruments; DIAGNOSTIC reagents &; test kits
- Publication
BMC Biotechnology, 2013, Vol 13, Issue 1, p1
- ISSN
1472-6750
- Publication type
Article
- DOI
10.1186/1472-6750-13-104