Nextra xt kit

The metagenomic DNA were processed to prepare the 16S rRNA libraries. Amplicon libraries were prepared using the Nextra XT kit (Illumina Inc., San Diego, United States). Archaea specific primers Arch-344F (Wemheuer et al., 2012 (link)) and Arch-806R (Takai and Horikoshi, 2000 (link)) were used for preparing initial amplicons. The PCR amplification was performed as follows: initial denaturation at 95 °C for 3 min followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min. A reaction without template was used as a negative control, whereas the Methanobrevibacter smithii DNA template was used as a positive control during the PCR amplification. After purification of the PCR products using AmPure XP beads Nextra XT index primers (i5 and i7) were added using Nextra XT index kit. All the indexed amplicon libraries were purified with AMPureXP beads (Beckman Coulter Life Sciences, United States) and analyzed individually on a 4,200 Tape Station (Agilent Technologies, United States). The libraries were multiplexed (10–20 pM of each) and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, United States) using MiSeq reagent kit v3, and 2 × 300 bp paired-end reads were generated to obtain approximately 0.1 million sequences per library.