Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota
Abstract
1. Introduction
1.1. Role of the Microbiome in Breast Cancer and the Importance of Microbiome Research
1.2. Sequencing Strategies and Their Significance in Microbiome Studies
1.3. Challenges in Microbiome Research and Rationale
2. Materials and Methods
2.1. Design
2.2. Biological Samples
2.3. DNA Isolation Methods
2.3.1. Sample Lysis
2.3.2. DNA Extraction
2.4. DNA Sequencing
2.5. Bioinformatic Analyses
2.5.1. 16S rRNA Method
2.5.2. Shotgun Method
2.6. Statistic Analysis
3. Results
3.1. Human DNA Depletion
3.2. 16S rRNA Sequencing
3.3. Shotgun
3.4. Diversity Index Comparison
3.5. Microbiome Balance
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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(A) | ||||||
Phylum | Healthy Women (Controls) | |||||
Feces | Breast | |||||
A (n = 5) | B (n = 5) | C (n = 5) | A (n = 5) | B (n = 5) | C (n = 5) | |
Actinomycetota | 4.7 (0.3–13.8) a | 7.8 (2.7–10.7) a | 14.4 (13.5–17.5) b | 5.7 (4.8–6.2) | 7.3 (5.6–9.9) | 6.6 (5.0–8.0) |
Bacteroidota | 6.3 (3.0–22.8) a | 27.2 (15.2–33.9) b | 6.6 (2.6–22.7) a | 16.6 (9.4–19.0) a | 15.9 (6.2–19.2) a | 8.9 (7.1–11.3) b |
Bacillota | 74.6 (69.7–87.0) a | 62.5 (48.7–72.9) b | 74.1 (63.1–79.6) ab | 57.5 (46.2–63.7) a | 64.6 (52.3–67.6) ab | 71.7 (60.0–78.6) b |
Fusobacteria | 0 (0–0.03) | 0 (0–0.2) | 0 (0–0) | 0 (0–0.04) | 0 (0–0.1) | 0 (0–0.01) |
Pseudomonadota | 2.0 (0.2–2.4) a | 0.6 (0.3–1.9) ab | 0.3 (0.1–1.3) b | 4.7 (3.6–10.0) a | 4.8 (3.7–12.6) a | 2.6 (0.5–2.8) b |
Verrucomicrobiota | 2.3 (0–6.8) a | 0 (0–26.5) b | 0.02 (0–8.0) b | 2.3 (0.8–3.7) | 3.0 (0.2–4.1) | 1.5 (1.1–3.1) |
Unassigned | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0.04 (0–0.3) | 0 (0–0.5) | 0.09 (0–0.2) |
(B) | ||||||
Phylum | Patients with Breast Cancer (Cases) | |||||
Feces | Breast | |||||
A (n = 5) | B (n = 5) | A (n = 5) | B (n = 5) | |||
Actinomycetota | 1.6 (1.1–29.0) | 5.0 (3.5–7.2) | 4.9 (1.9–6.0) | 3.1 (1.5–5.1) | ||
Bacteroidota | 9.6 (0.8–32.0) a | 17.3 (3.7–24.6) b | 18.3 (16.1–18.9) a | 19.4 (18.4–23.5) b | ||
Bacillota | 68.2 (52.8–95.0) | 72.4 (58.0–87.7) | 63.0 (57.7–70.2) | 66.9 (60.0–69.2) | ||
Fusobacteria | 0 (0–0.01) | 0 (0–0) | 0 (0–0.1) | 0.08 (0–0.3) | ||
Pseudomonadota | 2.0 (0.9–7.7) | 1.2 (0.3–7.6) | 4.6 (3.1–8.2) | 4.2 (2.5–9.1) | ||
Verrucomicrobiota | 0.9 (0–6.3) a | 5.2 (0.06–14.2) b | 2.3 (1.5–3.0) | 3.0 (1.9–3.5) | ||
Unassigned | 0 (0–0) | 0 (0–0) | 0.04 (0.02–0.2) | 0.01 (0–0.02) |
Variables | Control | Patients with Breast Cancer | ||||||
---|---|---|---|---|---|---|---|---|
Feces | Breast | Feces | Breast | |||||
A (n = 5) | B (n = 5) | A (n = 5) | B (n = 5) | A (n = 5) | B (n = 5) | A (n = 5) | B (n = 5) | |
Actinomycetota | 0 (0–21.8) | 1.9 (0–16.0) * | 0 (0–0) | 1.2 (1.0–1.0) | 0.3 (0–26.3) | 6.7 (2.0–14.0) * | 0 (0–0) | 0.7 (1.0–1.0) |
Bacteroidota | 0 (0–5.5) | 4.2 (0–21.0) * | 0 (0–0) | 10.4 (10.0–10.0) | 5.5 (0–39.2) | 7.7 (1.0–28.0) | 0 (0–0) | 13.4 (12–15) |
Bacillota | 88.3 (72.6–100) | 83.9 (63–100) | 0 (0–0) | 94.2 (88.0–100.0) | 77.5 (46.8–100) | 83.2 (56–100) | 0 (0–0) | 83.9 (80–88) |
Pseudomonadota | 0 (0–0) | 1.7 (0–3.0) * | 0 (0–0) | 0 (0–0) | 0 (0–6.7) | 2.7 (1.0–5.0) * | 0 (0–0) | 4.7 (5.0–5.0) |
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Plaza-Díaz, J.; Fernández, M.F.; García, F.; Chueca, N.; Fontana, L.; Álvarez-Mercado, A.I. Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota. Life 2025, 15, 599. https://doi.org/10.3390/life15040599
Plaza-Díaz J, Fernández MF, García F, Chueca N, Fontana L, Álvarez-Mercado AI. Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota. Life. 2025; 15(4):599. https://doi.org/10.3390/life15040599
Chicago/Turabian StylePlaza-Díaz, Julio, Mariana F. Fernández, Federico García, Natalia Chueca, Luis Fontana, and Ana I. Álvarez-Mercado. 2025. "Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota" Life 15, no. 4: 599. https://doi.org/10.3390/life15040599
APA StylePlaza-Díaz, J., Fernández, M. F., García, F., Chueca, N., Fontana, L., & Álvarez-Mercado, A. I. (2025). Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota. Life, 15(4), 599. https://doi.org/10.3390/life15040599