cave  metagenome
Extracted eDNA was purified by electroelution technique with slight modifications. Specifically, crude eDNA was separated through 1% agarose gel in 0.5X TBE buffer with constant voltage of 100 v. After 1h, any remaining DNAs that did not migrate into agarose gel were washed out of the loading wells, and the buffer was replaced. The voltage was then reduced to 20 v, and the gel was additionally run for 5h. Then, a slice of agarose gel containing high molecular weight DNA was then cut out with a sharp scalpel. The excised gel was then put into a dialysis bag (Spectra/Por, USA) filled with 0.25x TBE buffer. Next, the bag was immersed in a shallow layer of 0.25x TBE buffer in a horizontal electrophoresis tank in an orientation that was in line with the electrodes, with the current at 100 v for 90 min. The polarity of electric current was then reversed for 1 min to release any eDNA sticking to the wall of dialysis bag. The buffer with eluted eDNAwas transferred into a 15 ml tube. One volume of chloroform:isoamylalcohol (24:1) was added and mixed before centrifugation at 6,000 g for 10 min. The upper phase was transferred to a clean 15 ml tube. Eluted eDNA was then precipitated by adding 1/10 volume of 3M sodium acetate (pH 5.2) and 0.7 volume of isopropanol. After 1 hour of incubation at room temperature, eluted eDNA was pelleted by centrifugation at 13,000 g for 20 min. After washing with 70% ethanol (vol/vol), eDNA pellet was air-dried at room temperature and resuspended in 50 l of sterilized MiliQ water.