title: Improved workflows for high throughput library preparation using the transposome-based Nextera system.
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.
authors.affiliation: Wellcome Trust Centre for Human Genetics, OX3 7BN Oxford, UK. slamble@well.ox.ac.uk.
authors.forename: Sarah
authors.initials: S
authors.lastname: Lamble
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Batty
authors.forename: Moustafa
authors.initials: M
authors.lastname: Attar
authors.forename: David
authors.initials: D
authors.lastname: Buck
authors.forename: Rory
authors.initials: R
authors.lastname: Bowden
authors.forename: Gerton
authors.initials: G
authors.lastname: Lunter
authors.forename: Derrick
authors.initials: D
authors.lastname: Crook
authors.forename: Bassam
authors.initials: B
authors.lastname: El-Fahmawi
authors.forename: Paolo
authors.initials: P
authors.lastname: Piazza
mesh_terms: D057205:Automation, Laboratory; D016360:Clostridioides difficile / Q000235:genetics; D017931:DNA Primers / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D057188:Workflow
chemical_list: D017931:DNA Primers