ITS2-seq of fungal community: lake sediment
we amplified the internal transcribed spacer 2 (ITS2) region of the nuclear ribosome using the universal primers [gITS7F, 5 - GTGARTCATCGARTCTTTG-3 and ITS4R, 5-TCCTCCGCTTATTGATATGC- 3]. The PCR amplifications were carried out with the following steps: an initial denaturation at 94 C for 5 minutes, followed by 24 cycles consisting of 30 seconds of denaturation at 94 C, 30 seconds of annealing at 56 C, and 20 seconds of extension at 72 C, with a final extension at 72 C for 5 minutes. To assess the presence of contamination and validate the amplification process, negative controls (no-template controls, NTCs) were included in each PCR set. The PCR products were then analyzed using 1.5% agarose gel electrophoresis, purified using AMPure XP beads (Beckman, USA), and quantified with the Qubit dsDNA assay kit using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA). The controls (extraction blanks and NTCs) were analyzed to detect any potential contamination across the workflow, and no amplification or DNA was detected in these samples. We then normalized the barcoded PCR products of bacterial or fungal samples at equal molar concentration and sequenced them with 2250 bp paired-end on the Illumina HiSeq sequencing platform (Illumina Inc.).