Preserving Sample Integrity for Microbial Community Analysis

Aliquots were snap frozen and stored at −80 °C until further processing to maximize the integrity of samples and avoid degradation resulting from long-term storage at 4 °C [76 (link), 77 ]. Samples were thawed at 4 °C prior to treatments. ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA, USA) was used as both the internal standard and the external standard. As the internal standard, 6.50 μL Zymo community was spiked into 1 mL aggregate sample, following the criterion that DNA of the species with the highest abundance in the Zymo community approximates 1% of the total DNA in the aggregate sample [32 (link), 78 (link)]. As the external standard, aliquots of the Zymo community were run in parallel with aggregate samples throughout the workflow to assure its performance.