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 .