16S rRNA metagenomics: increasing efficiency by blocking amplification of contaminating sequences from a eukaryotic host

23 July 2017

16S rRNA sequencing is the most common approach used to identify microbes without the need to sequence entire genome. It is fast, cost-effective and flexible; however the method suffers some inefficiencies.

The major problem encountered, when investigating microbial communities associated with a eukaryotic host, is that >80% of 16S sequences can originate from the host’s genome, plastid or mitochondria.

To mitigate this contamination we introduced a new library preparation protocol that uses peptide nucleic acid (PNA) PCR clamps, i.e. synthetic oligomers that bind specifically to a unique signature in the contaminant sequence and physically block its amplification.

By applying a combination of PNAs that blocked both types of host 16S contamination in the same reaction we increased the yield of bacterial sequences for ~30% on three test samples derived from root microbiome (Figure1).

Figure 1. PNA clamps enrich bacterial sequences by specifically blocking amplification of contaminant sequences. 16S amplifications were performed in the absence and in the presence of PNA. Relative abundance of classified bacteria, plastid/mitochondria and other are shown.

 

In general, the use of PNAs had no effect on clustering of samples nor on relative abundances of bacterial families or OTUs, thus the enrichment of microbial sequences occurs without introducing any bias. What is more, it increased sequencing efficiency and reduced costs by enabling greater complexity of the microbial community to emerge at the lower depths.

Given that both the plastid and mitochondria PNA sequences are conserved among higher plants this approach is bound to function well for most plant microbiome projects.

Just try it!