Chloroplast sequence variation and the efficacy of peptide nucleic acids for blocking host amplification in plant microbiome studies
Why this work is in the frame
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Bibliographic record
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.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it