To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success
Abstract
1. Introduction
2. Materials and Methods
2.1. Plant Species
2.2. Initial Peptide-Nucleic Acid (PNA) Clamp Function Tests
2.3. PNA Clamp Metabarcoding
2.4. Metabarcoding Data Processing
3. Results
3.1. PNA Clamp Initial Verification
3.2. PNA Clamp Effects on Plant DNA Reads
3.3. Most Abundant ASVs with and Without PNA Clamps Added to Metabarcoding Library
3.4. PNA Clamp Effects on Alpha Diversity Measures
3.5. PNA Clamp Effects on Community Composition
3.6. Bacterial Species Associated with the Clamped Dataset
4. Discussion
4.1. Universal PNA Clamps Effectively Reduce Host Chloroplast, but Not Mitochondrial Reads
4.2. Addition of PNA Clamps Did Not Bias Bacterial Community Composition
4.3. Cost Comparison: Adding PNA Clamps Versus Increasing Read Depth per Sample
4.4. Proposed Decision Matrix for PNA Clamp Workflow and Verification
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
PCR | Polymerase chain reaction |
PNA | Peptide nucleic acid clamps |
ASV | Amplicon sequence variant |
mPNA | Mitochondrial peptide nucleic acid clamp |
pPNA | Chloroplast nucleic acid clamp |
WEHI | Walter and Eliza Hall Institute |
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(a) Without Peptide Nucleic Acid Clamps | (b) With Peptide Nucleic Acid Clamps | ||||
---|---|---|---|---|---|
Closest Match in SILVA SSU Database (Version 138) to ASV | Source | No. of Reads | Closest Match in SILVA SSU Database (Version 138) to ASV | Source | No. of Reads |
Themeda triandra | PC | 507,898 | Triticum aestivium | PM | 301,527 |
Eucalyptus sp. | PC | 292,468 | Eucalyptus grandis | PM | 232,199 |
Eucalyptus grandis | PM | 245,108 | Pantoea agglomerans | B | 225,290 |
Elymus sibiricus | PM | 190,490 | Pantoea ananatis | B | 209,750 |
Triticum aestivium | PM | 120,740 | Enterobacter hormaechei | B | 98,460 |
Pantoea agglomerans | B | 17,807 | Eucalyptus wimmerensis | PC | 84,243 |
Pantoea ananatis | B | 16,337 | Pseudomonas psychrotolerans | B | 69,391 |
Pseudomonas psychrotolerans | B | 9928 | Roseobacter sp. | B | 66,294 |
Pseudomonas sp. | B | 8478 | Eucalyptus grandis | PC | 50,592 |
Klebsiella pneumoniae | B | 5195 | Pseudomonas viridiflava | B | 24,588 |
Erwinia sp. | B | 2344 | Erwinia sp. | B | 23,535 |
Pelomonas aquatica | B | 2130 | Eucalyptus sp. | PC | 16,164 |
Altermonas oceani | B | 1681 | Klebsiella pneumoniae | B | 12,337 |
Qipengyuania sp. | B | 1248 | Themeda triandra | PC | 11,901 |
Luteibacter sp. | B | 1201 | Microlaena stipoides | PC | 11,768 |
Pseudomonas syringae | B | 1199 | Pseudomonas marginalis | B | 11,616 |
Sphingomonas sp. | B | 1143 | Luteibacter sp. | B | 11,118 |
Luteibacter sp. | B | 989 | Sediminibacterium ginsengisoli | B | 10,607 |
Microlaena stipoides | PC | 973 | Erythrobacter pelagi | B | 10,028 |
Psychromicrobium sp. | B | 892 | Pseudomonas versuta | B | 9512 |
Methylorubrum sp. | B | 847 | Paenibacillus nuruki | B | 9129 |
Pseudomonas sp. | B | 809 | Pseudomonas ovata | B | 8851 |
Dataset | Total No. of Bacterial Reads 1 | No. of ASVs 2 | Unique ASVs 2 | Percentage of Reads That Were Unique to Each Dataset | Shared ASVs 2 | Percentage of Reads That Were Shared in Each Dataset |
---|---|---|---|---|---|---|
Clamped | 615,442 | 363 | 284 | 14.39 | 79 | 85.60 |
Non-clamped | 161,633 | 132 | 53 | 0.33 | 79 | 99.67 |
Total | 777,075 | 495 | 337 | - | - | - |
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Mertin, A.A.; Blackall, L.L.; Brumley, D.R.; Liew, E.C.Y.; van der Merwe, M.M. To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success. Seeds 2025, 4, 28. https://doi.org/10.3390/seeds4030028
Mertin AA, Blackall LL, Brumley DR, Liew ECY, van der Merwe MM. To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success. Seeds. 2025; 4(3):28. https://doi.org/10.3390/seeds4030028
Chicago/Turabian StyleMertin, Allison A., Linda L. Blackall, Douglas R. Brumley, Edward C. Y. Liew, and Marlien M. van der Merwe. 2025. "To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success" Seeds 4, no. 3: 28. https://doi.org/10.3390/seeds4030028
APA StyleMertin, A. A., Blackall, L. L., Brumley, D. R., Liew, E. C. Y., & van der Merwe, M. M. (2025). To Clamp or Not to Clamp: Enhancing Seed Endophyte Metabarcoding Success. Seeds, 4(3), 28. https://doi.org/10.3390/seeds4030028