Peptide nucleic acid (PNA) clamps reduce amplification of host chloroplast and mitochondria rRNA gene sequences and increase detected diversity in 16S rRNA gene profiling analysis of oak-associated microbiota

Abstract

Chloroplast sequence variation and the efficacy of peptide nucleic acids for blocking host amplification in plant microbiome studies

Abstract

Background

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 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.

Results

Using 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.

Conclusions

When 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.

To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success

Abstract

Seed microbes play crucial roles in plant health, but studying their diversity is challenging due to host DNA contamination. This study aimed to optimise methodologies for investigating seed microbiomes across diverse plant species, focusing on the efficacy of peptide nucleic acid (PNA) clamps to reduce host DNA amplification. We tested PNA clamps on three plant species: Melaleuca quinquenervia (tree), Microlaena stipoides, and Themeda triandra (grasses). The effectiveness of PNA clamps was assessed through in silico analysis, axenic tissue culture, and metabarcoding techniques. In silico analysis confirmed the specificity of PNA clamps to the 16S rRNA gene V4 region of chloroplasts in the grass species. Axenic tissue culture experiments showed that applying PNA clamps at both 1 µM and 0.25 µM concentrations significantly reduced plant DNA amplification. Metabarcoding analyses further confirmed that PNA clamps effectively suppressed host DNA, enhancing microbial diversity estimates across all three species while preserving core microbial taxa. The efficacy of the clamps varied among host species, with T. triandra exhibiting the highest blocking efficacy, and chloroplast clamps outperforming mitochondrial ones. This study demonstrates that PNA clamps are a useful for improving seed endophyte metabarcoding datasets, although they require optimisation for some plant species. This knowledge will contribute to enhancing our understanding of seed microbiome diversity and its ecological implications.

Identifying the plant-associated microbiome across aquatic and terrestrial environments: the effects of amplification method on taxa discovery

Abstract

Plants in terrestrial and aquatic environments contain a diverse microbiome. Yet, the chloroplast and mitochondria organelles of the plant eukaryotic cell originate from free-living cyanobacteria and Rickettsiales. This represents a challenge for sequencing the plant microbiome with universal primers, as ~99% of 16S rRNA sequences may consist of chloroplast and mitochondrial sequences. Peptide nucleic acid clamps offer a potential solution by blocking amplification of host-associated sequences. We assessed the efficacy of chloroplast and mitochondria-blocking clamps against a range of microbial taxa from soil, freshwater and marine environments. While we found that the mitochondrial blocking clamps appear to be a robust method for assessing animal-associated microbiota, Proteobacterial 16S rRNA binds to the chloroplast-blocking clamp, resulting in a strong sequencing bias against this group. We attribute this bias to a conserved 14-bp sequence in the Proteobacteria that matches the 17-bp chloroplast-blocking clamp sequence. By scanning the Greengenes database, we provide a reference list of nearly 1500 taxa that contain this 14-bp sequence, including 48 families such as the Rhodobacteraceae, Phyllobacteriaceae, Rhizobiaceae, Kiloniellaceae and Caulobacteraceae. To determine where these taxa are found in nature, we mapped this taxa reference list against the Earth Microbiome Project database. These taxa are abundant in a variety of environments, particularly aquatic and semiaquatic freshwater and marine habitats. To facilitate informed decisions on effective use of organelle-blocking clamps, we provide a searchable database of microbial taxa in the Greengenes and Silva databases matching various n-mer oligonucleotides of each PNA sequence.

Peptide nucleic acid (PNA) clamps enhance root microbiome profiling in wheat and maize

Abstract

Background

Peptide Nucleic Acid (PNA) clamps represent a crucial molecular tool for reducing host DNA contamination during plant tissue microbiome profiling. This is particularly important when there is sequence similarity between the host organellar DNA (e.g., mitochondrial) and the targeted PCR sequences. However, the effectiveness and optimal concentration of universal PNA clamps can vary between plant species, necessitating a case-by-case evaluation. Here, we assessed the effectiveness of five concentrations (0.0, 0.25, 1.0, 2.0 and 4.0 µM) of mitochondrial and chloroplast PNA blockers (mPNA and pPNA) in reducing the amplification of organellar DNA and enhancing the profiling of prokaryotic communities across root tissues from 34 maize and 27 wheat samples cultivated under various soil and climatic conditions.

Results

We observed that host plant contamination in root samples was consistently high, with an average rate exceeding 95% across all samples. The application of PNA clamps significantly reduced plant host contamination by 2.4–27.2 times in a concentration-dependent manner. This reduction was more pronounced in maize samples than in wheat samples, particularly at lower doses (PNA ≤ 1.0 µM). PNA clamps also increased the read abundance of more than half of the observed microbiome phyla in the root tissues. The most substantial increase in prokaryotic read abundance was observed at a PNA concentration of 1.0 µM, without introducing significant bias to the prokaryotic community.

Conclusions

In conclusion, the introduction of universal PNA clamps during PCR assays significantly reduced amplification of host contamination and enhanced the detection of low-abundance microbiome and the depth of microbial profiling in both maize and wheat root tissues, with effects being concentration- and crop-specific.