Amplicon Sequencing Library Preparation

Amplicons were purified by Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, USA) and quantified by Qubit dsDNA HS Kit (Invitrogen) using a Qubit Fluorometer 3.0 (Invitrogen). Purified amplicons with a dsDNA concentration of at least 1.89 ng/μL (12nM) were diluted to 4 nM in 10 mM Tris, pH 8.5, and then pooled in equal volumes to create the sequencing library. The library was sequenced using a 12 pM loading concentration and MiSeq v3 Kit (Illumina, California, USA) on a MiSeq platform (Illumina) following manufacturer’s protocols. The Generate FASTQ workflow, paired‐end sequencing, and custom sequencing primers were used as previously described (28 (link), 33 (link)) to a target depth of >2000 reads per amplicon per sample.