Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics
Abstract
:1. Introduction
2. Results
2.1. Manual vs. Automatic DNA from FFPE Tissue Extraction
2.2. NGS Automatic Performance Workflow
3. Discussion
4. Materials and Methods
4.1. DNA Extraction Workflow
4.1.1. Biological Sample Collection and Macroscopic Evaluation
4.1.2. Manual DNA Isolation from FFPE Tissues
4.1.3. Automated DNA Isolation from FFPE Tissues
4.1.4. Quality and Quantity DNA Assessment
4.1.5. Assessment of PCR Amplifiable Fragment Length
4.1.6. Genomic DNA extraction
4.2. NGS Workflow
4.2.1. NGS Library Preparation, Quality and Quantity Assessment
4.2.2. NGS Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample | Extraction Method | NanoDrop (DNA, ng/µL) | Total DNA (ng) NanoDrop | 260/280 Ratio | Qubit (DNA, ng/µL) | Total DNA (ng) Qubit |
---|---|---|---|---|---|---|
24N | MM | 35.9 | 2513 | 1.83 | 12.4 | 868 |
24T | MM | 126.4 | 8848 | 1.74 | 26 | 1820 |
48N | MM | 64.8 | 4536 | 1.74 | 30.4 | 2128 |
48T | MM | 103 | 7210 | 1.78 | 36.6 | 2562 |
72N | MM | 58.9 | 4123 | 1.73 | 29 | 2030 |
72T | MM | 73 | 5110 | 1.76 | 37.8 | 2646 |
24N | PC | 171 | 3420 | 1.99 | 46.8 | 936 |
24T | PC | 623.6 | 12472 | 1.94 | 12.92 | 258.4 |
48N | PC | 156 | 3120 | 1.98 | 224 | 4480 |
48T | PC | 325.4 | 6508 | 1.98 | 154 | 3080 |
72N | PC | 546.6 | 10932 | 1.95 | 56 | 1120 |
72T | PC | 106.8 | 2136 | 2.07 | 4.6 | 92 |
24N | GR | 305.2 | 9156 | 1.95 | 74.4 | 2232 |
24T | GR | 647.4 | 19422 | 1.91 | 60.4 | 1812 |
48N | GR | 211.6 | 6348 | 1.93 | 244 | 7320 |
48T | GR | 393.6 | 11808 | 1.92 | 117.6 | 3528 |
72N | GR | 565.6 | 16968 | 1.9 | 112.8 | 3384 |
72T | GR | 194 | 5820 | 1.97 | 37.6 | 1128 |
Sample | DNA Extraction Method | DNA (ng/µL) Nanodrop | DNA (ng) Nanodrop | 260/280 Ratio | DNA (ng/µL) Qubit | DNA (ng) Qubit |
---|---|---|---|---|---|---|
24N | MM-O | 65.6 | 4920 | 1.6 | 8.94 | 670.5 |
24T | MM-O | 71.2 | 5340 | 1.62 | 8.48 | 636 |
48N | MM-O | 48.7 | 3648.8 | 1.63 | 4.28 | 321 |
48T | MM-O | 229.2 | 17190 | 1.84 | 77.2 | 5790 |
72N | MM-O | 297.4 | 22305 | 1.8 | 110 | 8250 |
72T | MM-O | 75.7 | 56738 | 1.71 | 16.6 | 1245 |
24N | MM-KF | 43.1 | 2155 | 1.7 | 8.34 | 417 |
24T | MM-KF | 92.6 | 4630 | 1.75 | 30.8 | 1540 |
48N | MM-KF | 51.6 | 2580 | 1.67 | 13.5 | 675 |
48T | MM-KF | 66.7 | 3335 | 1.71 | 23.6 | 1180 |
72N | MM-KF | 73.1 | 3655 | 1.75 | 24 | 1200 |
72T | MM-KF | 22.8 | 1140 | 1.51 | 5.76 | 288 |
Sample | Extraction Method | DNA (ng/µL) NanoDrop | DNA (ng) NanoDrop | 260/280 | DNA (ng/µL) Qubit | DNA (ng) Qubit |
---|---|---|---|---|---|---|
24N | MM-O | 65.6 | 4920 | 1.6 | 8.94 | 670.5 |
24T | MM-O | 71.2 | 5340 | 1.62 | 8.48 | 636 |
48N | MM-O | 48.7 | 3648.8 | 1.63 | 4.28 | 321 |
48T | MM-O | 229.2 | 17190 | 1.84 | 77.2 | 5790 |
72N | MM-O | 297.4 | 22305 | 1.8 | 110 | 8250 |
72T | MM-O | 75.7 | 5673,8 | 1.71 | 16.6 | 1245 |
24N | MM-KF | 43.1 | 2155 | 1.7 | 8.34 | 417 |
24T | MM-KF | 92.6 | 4630 | 1.75 | 30.8 | 1540 |
48N | MM-KF | 51.6 | 2580 | 1.67 | 13.5 | 675 |
48T | MM-KF | 66.7 | 3335 | 1.71 | 23.6 | 1180 |
72N | MM-KF | 73.1 | 3655 | 1.75 | 24 | 1200 |
72T | MM-KF | 22.8 | 1140 | 1.51 | 5.76 | 288 |
Sample | DNA Extraction Method | DIN Value | QC Score |
---|---|---|---|
24N | MM | 5.5 | −0.04 |
24T | MM | 5.9 | −0.04 |
48N | MM | 5.4 | −0.04 |
48T | MM | 5.6 | 0.15 |
72N | MM | 5.7 | −0.04 |
72T | MM | 5.9 | −0.04 |
24N | FC | 6.1 | −0.03 |
24T | FC | nv | −0.04 |
48N | FC | 1.9 | −0.02 |
48T | FC | 5.8 | 0.01 |
72N | FC | 5.7 | 0.01 |
72T | FC | nv | −0.01 |
24N | GR | 5.3 | 0.12 |
24T | GR | 5.7 | −0.03 |
48N | GR | 4.2 | −0.02 |
48T | GR | 5.5 | −0.01 |
72N | GR | 5.7 | −0.01 |
72T | GR | 4.5 | −0.01 |
24N | MM-O | 5.6 | −0.04 |
24T | MM-O | 5.1 | −0.04 |
48N | MM-O | 4.4 | −0.04 |
48T | MM-O | 6.2 | −0.03 |
72N | MM-O | 6.2 | −0.04 |
72T | MM-O | 6.2 | −0.02 |
24N | MM-KF | 4.7 | 0.01 |
24T | MM-KF | 5.8 | 0.00 |
48N | MM-KF | 4.8 | 0.03 |
48T | MM-KF | 4.8 | 0.03 |
72N | MM-KF | 4.8 | 0.04 |
72T | MM-KF | 4.1 | 0.03 |
NGS Variables | Automated | Manual |
---|---|---|
Cluster passing filter | 92% | 85.9% |
Q30 score | 85 | 80 |
Error rate | 0.15% | 0.18% |
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Share and Cite
Muscarella, L.A.; Fabrizio, F.P.; De Bonis, M.; Mancini, M.T.; Balsamo, T.; Graziano, P.; Centra, F.; Sparaneo, A.; Trombetta, D.; Bonfitto, A.; et al. Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics. Cancers 2019, 11, 1691. https://doi.org/10.3390/cancers11111691
Muscarella LA, Fabrizio FP, De Bonis M, Mancini MT, Balsamo T, Graziano P, Centra F, Sparaneo A, Trombetta D, Bonfitto A, et al. Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics. Cancers. 2019; 11(11):1691. https://doi.org/10.3390/cancers11111691
Chicago/Turabian StyleMuscarella, Lucia Anna, Federico Pio Fabrizio, Maria De Bonis, Maria Teresa Mancini, Teresa Balsamo, Paolo Graziano, Flavia Centra, Angelo Sparaneo, Domenico Trombetta, Antonio Bonfitto, and et al. 2019. "Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics" Cancers 11, no. 11: 1691. https://doi.org/10.3390/cancers11111691