Abstract
BackgroundThe ability to efficiently characterize microbial communities from host individuals can be limited by co-amplification of host organellar sequences (mitochondrial and/or plastid), which share a common ancestor and thus sequence similarity with extant bacterial lineages. One promising approach is the use of sequence-specific peptide nucleic acid (PNA) clamps, which bind to, and block amplification of, host-derived DNA. Universal PNA clamps have been proposed to block host plant-derived mitochondrial (mPNA) and plastid (pPNA) sequences at the V4 16S rRNA locus, but their efficacy across a wide range of host plant species has not been experimentally tested.ResultsUsing the universal PNA clamps, we amplified and sequenced root microbial communities from replicate individuals of 32 plant species with a most recent common ancestor inferred at 140 MYA. We found the average rate of host plastid contamination across plant species was 23%, however, particular lineages exhibited much higher rates (62–94%), with the highest levels of contamination occurring in the Asteraceae. We investigated chloroplast sequence variation at the V4 locus across 500 land plant species (Embryophyta) and found six lineages with mismatches between plastid and the universal pPNA sequence, including all species within the Asteraceae. Using a modified pPNA for the Asteraceae sequence, we found (1) host contamination in Asteraceae species was reduced from 65 to 23%; and (2) host contamination in non-Asteraceae species was increased from 12 to 69%. These results demonstrate that even single nucleotide mismatches can lead to drastic reductions in pPNA efficacy in blocking host amplification. Importantly, we found that pPNA type (universal or modified) had no effect on the detection of individual bacterial taxa, or estimates of within and between sample bacterial diversity, suggesting that our modification did not introduce bias against particular bacterial lineages.ConclusionsWhen high similarity exists between host organellar DNA and PCR target sequences, PNA clamps are an important molecular tool to reduce host contamination during amplification. Here, we provide a validated framework to modify universal PNA clamps to accommodate host variation in organellar sequences.
Highlights
The ability to efficiently characterize microbial communities from host individuals can be limited by co-amplification of host organellar sequences, which share a common ancestor and sequence similarity with extant bacterial lineages
We used the universal peptide nucleic acid (PNA) clamps designed by Lundberg et al [10] in a characterization of the root microbiomes of 32 flowering plant species from 14 families, with a most recent common ancestor inferred at 140 MYA
We investigated how rates of plastid contamination vary across host plant species, and whether these are associated with mismatches between universal plant-derived mitochondrial (mPNA) and plastid (pPNA) and target host organellar sequences
Summary
The ability to efficiently characterize microbial communities from host individuals can be limited by co-amplification of host organellar sequences (mitochondrial and/or plastid), which share a common ancestor and sequence similarity with extant bacterial lineages. Host-associated microbial communities are frequently characterized using deep amplicon sequencing [7], with bacterial taxa typically identified by their sequences at one or more of the hypervariable regions of the 16S rRNA gene [8] Amplification of these regions from host tissue can result in reduced sequencing efficiency due to host contamination, which is caused by sequence similarity due to the common ancestry shared between eukaryotic organelles and bacteria [9]. When sequencing the V4 region of the 16S rRNA gene from plant tissue, host-derived plastid and mitochondrial sequences can account for as much as 95% of all sequenced reads from a given sample [10] Such high-host contamination limits the number of samples that can be sequenced simultaneously with sufficient read depth, and quickly erodes the value of generation sequencing approaches in describing the microbiome. Techniques to limit host contamination are imperative to studies of host-associated microbiota
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