Miseq reagent kit v3 chemistry

The fungal barcoding region, i.e., the ITS rRNA gene region, was amplified as detailed by Guerreiro et al. (2018) (link). Briefly, library preparation comprised two sequential amplification steps. In the first PCR, the fungus specific primers ITS1F and ITS4 were used and modified at the 5′-ends to include sample-specific TAG sequences. In the second PCR, the sequencing primers, indices, and the P5 and P7 adapters for the Illumina sequencing were appended. Libraries were processed by the sequencing service of the Faculty of Biology at LMU Munich, and sequenced using an Illumina MiSeq® sequencer (Illumina, Inc., San Diego, CA, USA) with 2 × 250 bp paired end sequencing (MiSeq Reagent Kit v3 Chemistry, Illumina, Inc., San Diego, CA, USA).

Amplification of the V3–V4 hypervariable region of the 16S rRNA loci was performed using the primer set PCR1F_460 (5′-ACGGRAGGCAGCAG-3′) and PCR1R_460 (5′-TACCAGGGTATCTAATCCT-3′) (Andersson et al., 2008 (link); Liu et al., 2008 (link)). The 50-μl final-volume PCR1 reactions contained 5 × PCR buffer (Phusion), 10 mM of dNTP, 0.5 μM of each primer, 5 U of Taq Phusion, and 6 μl of genomic DNA. PCR conditions were as follows: one predenaturation step at 95°C for 5 min, 30 cycles of denaturation at 95°C for 20 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s, one post-elongation step at 72°C for 5 min, and then 4°C forever. PCR product quality and integrity were determined using 1% agarose gel electrophoresis. PCR product purification and secondary PCR amplification for the addition of the Illumina compatible sequencing adapters and unique per-sample indexes were conducted at GenoToul facility (Toulouse, France). Barcoded amplicons were quantified, quality-checked, normalized, pooled, and sequenced within two sequencing runs (May and November 2017) using the 2 × 250 paired-end method on an Illumina MiSeq instrument with a MiSeq Reagent Kit V3 chemistry (Illumina), according to the manufacturer’s recommendations.