Comparing DNA Extraction and 16S rRNA Gene Amplification Methods for Plant-Associated Bacterial Communities

Plant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16S ribosomal RNA (rRNA) gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially because the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16S rRNA gene amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct "fingerprint" of its own biases. Plant samples were less clear, with different species having different "best" options. For 16S rRNA gene amplification, we found that using peptide nucleic acid clamps provides the least taxonomic distortion, while chloroplastdiscriminating primers are easy and inexpensive but present significant bias in the results. Blocking oligos require a more complex protocol and optimization for each species and showed significant taxonomic distortion; therefore, we do not recommend them for off-the-shelf applications. Further methods development will hopefully result in protocols that are even more reliable and less biased than these.