Use of a filter cartridge combined with intra-cartridge bead-beating improves detection of microbial DNA from water samples

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Authors Masayuki Ushio
Journal/Conference Name Methods in Ecology and Evolution
Paper Category , ,
Paper Abstract Microbial communities play an important role in driving the dynamics of aquatic ecosystems. As difficulties in DNA sequencing faced by microbial ecologists are continuously being reduced, sample collection methods and DNA extraction protocols are becoming more critical to the outcome of any sequencing study. In the present study, I added a manual, intra-cartridge, bead-beating step in the protocol using a DNeasy® Blood & Tissue kit for DNA extraction from a filter cartridge without breaking the cartridge unit (“Beads” method) and compared its performance with those of two other protocols (“NoBeads” method, which was similar to the Beads method but without the bead-beating step and “PowerSoil” method, which followed the manual of the DNeasy® PowerSoil kit after breaking apart the filter cartridge). Water samples were collected from lake, river, pond and coastal ecosystems in Japan and DNA was extracted using the three protocols. Then, the V4 region of prokaryotic 16S rRNA genes was amplified. In the library preparation process, internal standard DNAs were included to estimate the number of DNA copies. The DNA library was sequenced using Illumina MiSeq and sequences were analysed using the amplicon sequence variant (ASV) approach. I found that, (a) the total prokaryotic DNA yields were highest with the Beads method, (b) the number of ASVs (a proxy for species richness) was also highest with the Beads method, (c) overall community compositions were significantly different among the three methods and (d) the number of method-specific ASVs was highest with the Beads method. These results were generally robust across samples from all aquatic ecosystems examined. 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 and it can reduce the potential contamination risk from the ambient air and/or other samples. Therefore, this method has the potential to become one of the major choices when one aims to extract aquatic microbial DNAs.
Date of publication 2019
Code Programming Language R

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