期刊
BMC BIOTECHNOLOGY
卷 13, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/1472-6750-13-104
关键词
Nextera; High-throughput; Library preparation; Sequencing; Normalisation
资金
- MRC [G0900747 91070]
- Wellcome Trust [090532/Z/09/Z]
- BBSRC [BB/I02593X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/I02593X/1] Funding Source: researchfish
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.
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