454 gs junior sequencing system

The microbial community structures in the water from PX-1 and PX-2 sampled after the M_W 3.2 earthquake (PX-1–17 and PX-2–17) and the M_W 5.5 earthquakes in August 2018 (PX-1–18 and PX-2–18) and April 2019 (PX-1–19 and PX-2–19) were analyzed via 16S rRNA gene-based pyrosequencing. Water samples were filtered via a 0.2 μm filter in the field and stored in a refrigerator (– 70 °C). Subsequently, DNA was extracted via a Fast DNA spin Kit (Qbiogen, USA). The extracted DNA was amplified via forward and inverse primers to distinguish each sample^{41 (link),42 (link)}. Pyrosequencing was conducted via a 454 GS junior sequencing system (Roche, NJ, USA) by Chun Laboratory (Seoul, Korea). Operational taxonomic units (OTUs) were used to determine bacterial community structures and calculate the abundance-based coverage estimator (ACE), Chao 1 richness estimator, Shannon and Simpson diversity indices, and rarefaction curves. Each sequence was analyzed and compared with sequences in the EzTaxon-extended database (Chun Lab, eztaxon-e.org) via BLASTIN searches and pairwise similarity comparisons. The full sequences were analyzed and compared with other known sequences that were available in the NCBI (National Center for Biotechnology Information) database.

DNA was extracted from the samples of sponge tissue (0.1–0.2 g) and primmorphs after bead beating using the TRIzol LS reagent (Invitrogen, USA) according to the manufacturer’s protocols. Total DNA from three technical replicates for each sample was suspended in 18 μl of RNase-free water and stored at −70 °C pending further analysis. The universal bacterial primers 518F and 1064R (Ghyselinck et al., 2013 (link)) were used to amplify the V4–V6 hypervariable region of the bacterial 16S rRNA gene. The following program was used to amplify 16S rRNA genes using PCR: 3 min at 96 ^∘C; 30 cycles at 94 °C for 20 s, 55 °C for 20 s and 72 °C for 1 min with a final 10-min incubation at 55 °C. PCR products were quantified using the NanoDrop device, mixed equally and sequencing using the 454 GS Junior Sequencing System (Roche, Basel, Switzerland) with GS FLX Titanium series reagents. Raw sequencing data are available in the NCBI Sequence Read Archive under accession number PRJNA480187.