PCR and purification
Primer sequences were designed for two Rlt housekeeping genes, recombinase A (recA ) and RNA polymerase B (rpoB ), and for two Rlt specific symbiosis genes, nodA and nodD(Additional File 1: Table S1 ).
The three primers are a target-gene forward inner primer, a universal forward outer primer, and a target-gene reverse primer. The concentration of the inner forward primer was 100-fold lower than the universal forward outer primer and the reverse primer (Figure 1 ) in order to reduce the competitiveness of this primer compared to the outer primer. The inner primer is essential for the first round of amplification, but its participation is undesirable in later rounds as it would assign a new unique UMI to an existing amplicon. The PCR reaction mixture and thermocycler programme are provided (Additional File 1: Tables S2 and S3 ).
PCRs were undertaken individually for each primer set using Platinum Taq DNA polymerase (Thermofisher Scientific Inc., USA) (Additional File 1: Table S2 ) and subsequently pooled and purified using AMPure XP Beads following the manufacturer’s instructions (Additional File 1: Table S5 ) (Beckman Coulter, USA). Successful PCR amplification was confirmed by running a 0.5X TBE 2% agarose gel at 90V for 2 hours.
For the DNA mixture samples, PCRs were run in triplicate. DNA from single strains was also processed as a control to determine the level of cross contamination between samples. Some samples were also amplified using Phusion High-Fidelity polymerase (Thermofisher Scientific Inc., USA), to evaluate whether use of a proof-reading polymerase improved the quality of the results using the PCR program described inAdditional File 1: Table S2 and Table S4 .