Dneasy powersoil kit

ZymoBIOMICS microbial community standards (Zymo Research) were used in this study to quantitatively evaluate the efficiency of direct PCR methods and the DNeasy PowerSoil kit. This microbial community standard is a defined composition comprising 5 Gram-positive bacteria (Listeria monocytogenes, Bacillus subtilis, Lactobacillus fermentum, Enterococcus faecalis, and Staphylococcus aureus), 3 Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica), and 2 yeast strains (Saccharomyces cerevisiae and Cryptococcus neoformans). The defined composition is well controlled and reported by the manufacturer as the real composition of the community. The ZymoBIOMICS microbial community standards were stored at −80°C until use. To avoid the effects of glycerol on microbial community standard cell lysis, 40 μl of the standard was thawed and centrifuged at 10,000 × g for 5 min. The supernatant was discarded, and the pellet was resuspended in 80 μl Milli-Q DNase-free water immediately before direct PCR or gDNA extraction.

Combined cells with rock or sediment substrate were pelleted through centrifugation at 13,000 r.p.m. for 3 min. For amplicon sequencing, unless specified in Supplementary Table 6, DNA was extracted using the Qiagen DNeasy PowerSoil kit (catalogue no. 47014) according to the manufacturer’s instructions as described previously^{48 (link)} with a minor modification, where mechanical shearing was carried out using the MP Biomedicals FastPrep-24 system (catalogue no. 116004500) at level 5.5 for 45 s. For genomic sequencing, incubated rock and sediment cultures were extracted using multiple approaches, including the Qiagen DNeasy PowerSoil kit, ZymoBIOMICS 96 MagBead DNA Kit (catalogue no. D4302; Zymo Research Corporation), Quick-DNA 96 Kit (catalogue no. D3010; Zymo Research Corporation), ZymoBIOMICS DNA Microprep Kit (catalogue no. D4301; Zymo Research Corporation) and a standard phenol/chloroform-based protocol. The list of samples and their extraction methods are provided in Supplementary Table 6.