EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strain
2.2. Sample Collection and Microbial Cells Extraction
2.3. EpicPCR
2.3.1. Cell Enumeration and Bead Formation
2.3.2. Fusion, Nested and Blocking PCRs and Sequencing
2.4. 16S rRNA Gene Amplification and Sequencing
2.5. Sequencing Data Analysis
3. Results and Discussion
3.1. Elaboration of Polyacrylamide Beads Carrying Isolated Environmental Cells
3.2. Checking epicPCR Design on Pure Culture
3.3. Tuning the Use of BP to Maximize epicPCR Efficiency on Environmental Cells
3.4. Exploring the Way to Validate OTUs in epicPCR Experiments
3.5. Plotting epicPCR Results on a Gold-Standard 16S-rDNA Tree
3.6. Synthetic Comparison of epicPCR and epicPCR 2.0 Methodologies
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characteristics | epicPCR | epicPCR 2.0 |
---|---|---|
Polyacrylamide beads formation | Done once per sample | Done three times in parallel per samples |
Beads formation quality check (by microscopy after cell staining) | More than 90% of beads are empty | More than 90% of beads are empty and 85% of non-empty beads only carry one cell |
Replicate strategy for performing the fusion-PCR | Triplicated amplification using a single beads batch per sample as template (=technical triplicates) | Single non-replicated amplification performed on each of the 3 beads batches (=biological triplicates) |
Fusion-PCR 1,2 | Performed with primers F1, R1-F2′ and R2 used at 1, 0.01 and 1 µM, respectively | |
Step between the fusion- and the nested-PCRs | None | Additional “blocking PCR” step using the blocking primers at 3.2 µM alone |
Nested-PCR2 | Performed with the primers F3 + SA and R3 + SA at 0.3 µM and blocking primers at 3.2 µM | Performed with the primers F3 + SA and R3 + SA at 0.3 µM and blocking primers at 0.32 µM |
Minimum of reads for creating an OTU in each replicate | 100 | 10 |
Preferential OTU validation method | OTU detected in each of the 3 technical replicates | OTU detected in each of the 3 biological replicates |
Confidence levels for characterizing OTU validation | None | Yes; from 5, the best one, to 1 |
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Roman, V.L.; Merlin, C.; Virta, M.P.J.; Bellanger, X. EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach. Microorganisms 2021, 9, 1649. https://doi.org/10.3390/microorganisms9081649
Roman VL, Merlin C, Virta MPJ, Bellanger X. EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach. Microorganisms. 2021; 9(8):1649. https://doi.org/10.3390/microorganisms9081649
Chicago/Turabian StyleRoman, Véronica L., Christophe Merlin, Marko P. J. Virta, and Xavier Bellanger. 2021. "EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach" Microorganisms 9, no. 8: 1649. https://doi.org/10.3390/microorganisms9081649