High fidelity sbfi

A pilot test was first carried out with three strains (AOB325, AOB413, AOB504). From this data set a minimum number of reads and number of samples that can be multiplexed was determined. Genomic DNA was digested with high‐fidelity SbfI (New England Biolabs), applying a RAD library preparation protocol modified from Amores et al. (2011 (link)) and Etter et al. (2011 (link)). Modifications (according to Rengefors et al., 2021 (link)) included an increased amount of ligase (2000 U µl^–1 T4 ligase) and decreased volume of NEB2 buffer (1 µl). In addition, AMPure XP beads were used to remove redundant P1 adapters and elution was done in three steps to increase the DNA yield, following the repair end and overhang addition step. AMPure XP beads were also used instead of column purification after the P2 adapter ligation. The final full amplification was performed with 16 µl template and 16 PCR cycles. After P1 adapter ligation, 20 uniquely barcoded (6 bp) strains were multiplexed per lane. Samples were subsequently sequenced on a HiSeq2500 system (Illumina), using paired‐end 125‐bp read length and v4 sequencing chemistry. Raw sequence reads are deposited in the European Nucleotide Archive (BioProject PRJEB41677).