Zymo zr 96 dna clean and concentrator 5 kit

PCR amplicons were purified using the Zymo ZR-96 DNA Clean and Concentrator-5 Kit (Zymo Research, catalog number D4024) according to the manufacturer’s instructions. The purified products were quantified using Quant-iT dsDNA Assay Kit, High Sensitivity (Thermo Fisher, catalog number Q33120) according to the manufacturer’s instructions. The quantified PCR amplicons were normalized by diluting in EB buffer to achieve equimolar concentrations of 1 ng/µL each across all samples and thereafter mixed to create amplicon pools of nonoverlapping MIDs. Sequence library preparation of the amplicon pools was performed according to the manufacturer’s instructions using the KAPA HyperPrep Kit (Roche, catalog number KK8504) with dual index adapters (Roche, catalog number 08278555702). Successful library preparation was confirmed using a random sample of 20 libraries on the Agilent High Sensitivity D1000 ScreenTape System (Agilent, catalog number 5067-5584). Both the ama1 and mdr1 adapter-ligated amplicon libraries were then quantified, normalized to 1 ng each, and mixed to create 1 final pool. Paired-end sequencing (2 × 300 bp chemistry) of the final pool was performed on the Illumina MiSeq platform using the MiSeq Reagent Kit v3 (Illumina, catalog MS-102-3003 number) at the University of North Carolina at Chapel Hill high-throughput sequencing facility.