D2 metabarcoding sequencing of Aviator X Pro fungicide treated Barley cultivar "Granger"
The concentration of amplicons in each MID tagged sample was evaluated based on qPCR RN and CT values to construct approximately equimolar mini-pools. Mini-pool concentration was then assessed using the QIAxcel fragment analyser (QIAGEN, Netherlands) and samples were pooled accordingly to build a single sequencing library representing DNA from each sample. Fragments were size-selected for 250600 bp lengths using the Pippin Prep instrument (Sage Sciences; USA). The library was purified using the QIAquick PCR purification kit (QIAGEN, Netherlands), yielding 50 L of library. The DNA concentration of the purified library was determined using the Qubit Fluorometric Quantitation instrument (Thermo Fisher Scientific, USA) and diluted to 2 M DNA. Lastly, the library was spiked with PhiX Control v2 library (Illumina, USA) to increase sequence diversity within the library.  Paired-end sequencing was carried out on the Illumina MiSeq platform (Illumina, USA) (2 x 260 bp sequencing cycles) using the standard flow cell and MiSeq Reagent Kit v2 (Illumina, USA) according to the manufacturers instructions, except with custom forward and reverse sequencing primers. Paired end reads were merged together using the StitchReads command in MiSeq Reporter v2.3, with a minimum overlap of 10 base pairs.