title: Illumina MiSeq paired end sequencing
design: Amplicon sequencing of rhizospheric soil attached to plants imported to Norway targeting the ITS1 region with primers specific for oomycetes. Samples were sequenced before and after enrichment by baiting.
library_construction_protocol: From each 1 litre soil sample, 50 mL were sub-sampled for metabarcode analysis before and after enrichment. The sub-samples were homogenized, and total eDNA was isolated from 250 mg soil per sub-sample using the DNeasy PowerSoil kit (Qiagen). The 250 mg fractions of each sub-sample were chemically lysed in buffer C1 (DNeasy PowerSoil kit) for 10 minutes before mechanical homogenization using beads in a FastPrep-24 homogenizer (MP Biomedicals) for 2 x 45 s. After homogenization and centrifugation, DNA isolation from the supernatant was automatized in a QIAcube (Qiagen) using the manufacturerâ€™s PowerSoil protocol. Indexed libraries for paired-end sequencing of the ITS1 metabarcoding region were generated using a one-step PCR with indexed Oomycete specific primers taken from Agler et al. (2016). After amplification, the DNA concentrations of the PCR products were measured using a Qubit 2.0 Fluorometer (Invitrogen), and 100 ng DNA per sample were used for library generation. The libraries were purified twice using the MinElute PCR Purification Kit (Qiagen), the DNA concentration measured again using the Qubit Fluorometer, and integrity of the DNA was confirmed in the 2100 Bioanalyzer (Agilent). Sequencing was performed on an Illumina MiSeq System using MiSeq Reagent Kit v3 (600-cycle) chemistry (Illumina), with 15 and 20 pM of the libraries in two separate runs together with 10 % PhiX control and sequencing primers.