Microbial Community Profiling by Metabarcoding

The archaeal 16S, eukaryotic 18S and fungal ITS1 genes were PCR amplified using primers listed in Supplementary Table 1. The resulting PCR products were purified using Agencourt AMPure XP DNA purification Bead (Beckman Coulter, USA) and quantified using Nanodrop-1000 (Thermo Scientific, USA). Then, PCR products of all NGTs samples (n = 19), all New-DMs (n = 14) and all Known-DMs (n = 16) were pooled by mixing equal quantities of concentration normalized PCR products. This way we obtained three pools for each group, NGTs, New-DMs and Known-DMs for archaeal 16S rRNA, eukaryotic 18S rRNA and fungal ITS1 genes. All the pooled samples were then sequenced using Ion Torrent PGM. Since fungal ITS amplicons varied in length, we fragmented 100 ng of it with Ion Shear Enzyme mix (Ion Xpress Plus Fragment Library preparation kit, Life Technologies) for 20 min and 200 bp size fragments were selected before adapter ligation step (Tang et al., 2015 (link)).