Preparation of library for NGS
For the rhizosphere and riparian soil samples, a nested polymerase chain reaction (PCR) was performed to amplify a nif D-K intergenic spacer (IGS) region in the samples. We adopted nested PCR to collect a sufficient amount of Frankia DNA for NGS (Ben Tekaya et al., 2018; Rodriguez et al., 2016). An approximately 500-bp fragment of thenif D-K IGS region was amplified by the PCR approach with theFrankia -specific primer pair, nif D1310frGC and nifKR331frGC (Kagiya and Utsumi, 2020). PCR amplification was performed as follows: one cycle at 95 °C for 2 min, followed by 35 cycles at 95 °C for 1 min and 64 °C for 5min, and a final step of one cycle at 72 °C for 5 min. The PCR products of soil samples and extracted DNA from host nodules were subjected to the same PCR method. PCR was performed to amplify an approximately 350-bp fragment of the nif D-K IGS region with the Frankia -specific primer pair (Table S1). This process was performed using the same method as the first-stage PCR for the usual NGS library preparation workflow. PCR amplification was performed as follows: one cycle at 95 °C for 2 min, followed by 35 cycles of 95 °C for 1 min and 61 °C for 5 min, and a final step of one cycle at 72 °C for 5 min. All successful PCR products were cleaned using AMPure XP magnetic beads (Beckman Coulter). These cleaned products were tagged by unique indices per sample performing index. Index PCR was performed as follows: one cycle at 95 °C for 3 min, followed by 8 cycles at 95 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec, and a final step of one cycle at 72 °C for 5 min. After the index PCR, the products were cleaned again with AMPure magnetic beads, and then libraries with the same amplicon concentration were pooled. These samples were analyzed, using Illumina MiSeq with paired-end 300 bp reads (Macrogen, Korea).