18S rRNA Gene Analysis by Illumina Sequencing

Partial 18S rRNA gene sequences (V4 region, ~380 bp) were amplified using primers Euk454F (5′-CCAGCASCYGCGGTAATTCC-3′) and EukR (5′-ACTTTCGTTCTTGATYRA-3′) (Logares et al., 2012). Polymerase chain reaction (PCR) mixture (20 μL) contained 1× KAPA Taq EXtra HotStart ReadyMix (Roche, Basel, Switzerland), 0.2 μM of each primer, and 1–5 μL template DNA. PCR was performed under the following cycling conditions: initial annealing at 95°C for 3 min, followed by 25–35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and with a final extension at 72°C for 5 min. All PCR products were purified with AMPure XP (Beckman Coulter). As some of the PCR products included multiband or smeared bands, after agarose gel electrophoresis, PCR products were excised from the gel and purified using a NucleoSpin Gel and PCR clean-up kit (Macherey Nagel, Germany). The PCR products were labeled with a sample-unique index and Illumina adapter sequences at their 5′ end using the Nextera XT index kit v2 (Illumina, San Diego, CA, the United States). The PCR mixture (10 μL) contained 1× KAPA HiFi HS ReadyMix, 2 μL each of forward and reverse primers, and 1 μL of the recovered PCR products. PCR was performed under the following cycling conditions: 95°C for 3 min, followed by 8 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and with a final extension at 72°C for 5 min. All index PCR products were purified with AMPure XP and measured using a Qubit 2.0 Fluorometer (ThermoFisher Scientific) with Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). Tagged amplicons were mixed with PhiX control DNA at a ratio of 80:20 and used as a template for MiSeq paired-end sequencing (2 × 300 bp) using Reagent kit v3 (Illumina) at the National Institute of Polar Research.