16S V5 V6 Metagenomics Kit Offers Enhanced Bacterial Identification in Plant Associated Samples

In the evolving field of bacterial metagenomics, many researchers choose to use variable regions of the 16S rRNA gene to understand which bacteria are present and how many bacteria are present in a sample. This approach can be compromised by contaminants in the sample which can amplify with the primers chosen. Bacterial profiling in plant samples can be difficult because of the homology between bacterial 16S rRNA and chloroplast 16S rRNA. The use of primers binding to 16S hypervariable regions V 1 – V4, which are traditionally used for bacterial profiling, amplify both the bacterial and plant 16S rRNA genes, skewing results. Here a methodology is presented for bacterial profiling of plant samples using degenerate primers that target the fifth and sixth variable regions of the 16S rRNA gene. In the presence of up to 90% Arabidopsis DNA, this method was still effective at identifying the 19 individual species of bacteria in the mock community. There were minimal reads and libraries formed by amplification of the Arabidopsis DNA. In comparison, when the same sample was enriched using 16S V4 specific primers, the vast majority of the reads mapped to the Arabidopsis chloroplast genome, and much information and representation of the bacterial community was lost. For generating sequencing libraries from samples which have the potential to be contaminated by plant or chloroplast DNA, a V5 V6 metagenomics kit is recommended.