S2 focused ultrasonicator

A composite ‘meta’-SCOBY was made by pooling equimolar amounts of DNA from each of the 103 individual Kombucha SCOBY DNA extracts. This synthetic representative sample was prepared for sequencing by CGRB using the TruSeq Nano PCR-free kit (Illumina, San Diego, CA, USA), with DNA shearing performed using the S2 focused ultrasonicator (Covaris, Woburn, MA, USA) as suggested in the Illumina protocol. Library quality was assessed using the Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) and a Qubit 2.0 fluorimeter (Invitrogen, Carlsbad, CA, USA). The meta-SCOBY library was sequenced using one Illumina HiSeq 3000 1 × 150 bp lane (Illumina, San Diego, CA, USA) by the CGRB.

A plant visibly infected with both Hyaloperonospora arabidopsidis and Albugo sp. was collected in Fall 2014 from the village of Gniebel (48° 34′ 34.10″ North Lat., 9° 10′ 55.42″ East Long.) using sterile tweezers and scissors, placed in a sterile 15 mL tube, and brought back to the lab on ice where it was frozen at −80 °C until further processing. The frozen plant was ground in the presence of liquid nitrogen using a mortar and pestle that was lined with four layers of autoclaved aluminum foil. Approximately 250 g of the resulting powder was used for DNA extraction, using a custom protocol we previously described [22 (link)]. Briefly, the sample was subjected to bead-beating in the presence of 1.5% sodium dodecyl sulfate (SDS) and 1 mm garnet rocks, followed by SDS cleanup with 1/3 volume 5 M potassium acetate, and then SPRI beads. The library was prepared using the TruSeq Nano kit (Illumina, San Diego, CA, USA), with DNA shearing performed with a S2 focused ultrasonicator (Covaris, Woburn, MA, USA) as suggested in the manufacturer’s protocol. Rather than Illumina adapters, we used custom adapters described in ref. [24 (link)]. The sample was sequenced on one lane of a HiSeq 2000 instrument (Illumina), using a 100 bp single-end kit.

Genomic DNA from Arabidopsis flowers and leaves was extracted using a DNeasy Plant Mini Kit (Qiagen). Concentration of the DNA was measured by Qubit dsDNA Broad-Range Assay kit (ThermoFisher). 50 μl aliquots containing 25, 50, 150, and 400 ng DNA were sheared by an S2 Focused-ultrasonicator (Covaris) to ~ 200 bp in average size using these parameters: intensity 5, duty cycle 10%, cycles per burst 200, treatment time 120 s.