Abstract
Microbial communities play an important role in driving the dynamics of aquatic ecosys-tems. As difficulties in DNA sequencing faced by microbial ecologists are continuously being reduced, sample collection methods and the choice of DNA extraction protocols are becoming more critical to the outcome of any sequencing study. In the present study, I added a manual bead-beating step in the protocol using a DNeasy® Blood & Tissue kit for DNA extraction from a filter cartridge (Sterivex™ filter cartridge) without breaking the cartridge unit (“Beads” method), and compared its performance with those of two other protocols that used the filter cartridge (“NoBeads” method, which was similar to the Beads method but without the bead-beating step, and “PowerSoil” method, which followed the manual of the PowerSoil DNA extraction kit after breaking apart the filter cartridge). Water samples were collected from lake and river ecosystems in Japan, and DNA was extracted using the three protocols. Then, the V4 region of prokaryotic 16S rRNA genes was amplified. In addition, internal standard DNAs were included in the DNA library preparation process to estimate the number of 16S rRNA gene copies. The DNA library was sequenced using Illumina MiSeq, and sequences were analyzed using the amplicon sequence variant (ASV) approach (DADA2). I found that, 1) the total DNA yields were highest with the Beads method, 2) the number of ASVs (a proxy for species richness) was also highest with the Beads method, 3) overall community compositions were significantly different among the three methods, and 4) the number of method-specific ASVs was highest with the Beads method. In conclusion, the inclusion of a bead-beating step performed inside the filter cartridge increased the DNA yield as well as the number of prokaryotic ASVs detected compared with the other two methods. Performing the bead-beating step inside the filter cartridge causes no dramatic increase in either handling time or processing cost. Therefore, this method has the potential to become one of the major choices when one aims to extract aquatic microbial DNAs.