Nextera indexing for multiplexing and MiSeq sequencing
Samples were indexed for multiplexed sequencing libraries with Nextera
XT DNA Library Preparation Kit v2 set A (Illumina, USA) using the
Phusion High-Fidelity DNA polymerase (Thermofisher Scientific Inc.,
USA). PCR reaction mixture and programme are detailed inAdditional File 1: Tables S6 and S7 Indices were added in
unique combinations as specified in the manufacturer’s instructions
(Illumina, USA).
The PCR product was purified on a 0.5X TBE 1.5% agarose gel and
extracted with the QIAQuick gel extraction kit (QIAGEN, USA) (expected
band length: ~454bp). PCR amplicon concentrations were
quantified using GelAnalyzer2010a and normalised to 10nM . A pooled
sample was quantified and checked for quality by Bioanalyzer (Agilent,
USA) before sequencing using Illumina MiSeq (2x300bp paired end reads)
by the University of York Technology Facility. A detailed protocol is
available in Additional File 1 .