Amplicon library preparation for Illumina sequencing

Sequencing libraries were generated by three rounds of PCR, according to previously published protocols^{9 (link)}. After primary PCR, a 5′ linker sequence was added during nested PCR. Nested PCR products were subject to another PCR round with primers binding to the linker sequences and carrying Illumina sequence adapters plus an eight nucleotide long sample-specific molecular index to permit pooling of amplicons for sequencing and later de-multiplexing. The final sequence library was purified with NucleoMag beads. Sequencing was performed on an Illumina MiSeq platform in paired-end mode (2 × 250 bp) using Illumina MiSeq reagent kit v2 (500-cycles) together with Enterobacteria phage PhiX control (Illumina, PhiXControl v3).