DNA extraction, library preparation, and sequencing
Nodule samples were thawed at ambient temperature and crushed using a sterile homogeniser stick. DNA was extracted from the homogenised nodule samples using the DNeasy PowerLyzer PowerSoil DNA isolation kit (QIAGEN, USA). DNA was amplified for two core genes (rpoB and recA ) and two accessory genes (nodA and nodD ). Amplification, library preparation, and sequencing was done using the MAUI-seq method . Sequencing was done on a Illumina MiSeq (2x300bp paired end reads) by the University of York Technology Facility.
The amplification and library preparation reported in this study were done at an early stage of method development, leading to some missing data (Figure 2 , nSamples_rpoB: 105, nSamples_recA: 153, nSamples_nodA: 129, nSamples_nodD: 130). The method has been improved since then and now has a robust and reliable amplification rate and sample recovery .