Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Information
2.2. MPS Library Preparation
2.3. Library Quantitation and Dilution
2.4. Emulsion PCR, Ion Chip Loading, and Sequencing
2.5. Data Analysis
3. Results
3.1. Hair Results
3.2. Bones (and Teeth) Results
3.3. FFPE Tissue Results
3.4. Nail and Buccal Swab Results
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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qPCR Sample Quantitation | ||||||
---|---|---|---|---|---|---|
Sample Name | Sample Type | Large Target (pg/µL) | Small Target (pg/µL) | mt Target (mtCopies/µL) | Legacy Results (Nuclear and Mitochondrial) | Precision ID Volume Used (µL) |
Hair Case 1—Item 1 | Hair—2 cm proximal end | 0 | Not Applicable (N/A) | 16,000 | No short tandem repeat (STR); hypervariable regions I/II (HVI/HVII) Mixture | 3.4 1 |
Hair Case 1—Item 2 | Hair Shaft—2 cm after item 1 sampling | 0 | N/A | 2700 | No STR; HVI/HVII Mixture | 2.8 1 |
Hair Case 1—Item 3 | Hair Shaft—2 cm after item 2 sampling | 0 | N/A | 160 | No STR; HVI/HVII Mixture | 3.3 1 |
Hair Case 2—Item 1 | Hair Root + Shaft | 0 | 0 | N/A | No STR; Partial HVI/HVII Results and a Mixture | 15 1 |
Hair Case 2—Item 2 | Hair Shaft—2 cm after root | 0 | 0 | N/A | No STR; Partial HVI/HVII Results and a Mixture | 15 1 |
Hair Case 3—Item 1 | Hair Shaft | N/A | N/A | N/A | No STR; No HVI/HVII Results | 15 1 |
Hair Case 3—Item 2 | 2nd Hair Shaft | N/A | N/A | N/A | No STR; No HVI/HVII Results | 15 1 |
Hair Case 4 | Hair Shaft | N/A | N/A | 3525 | HVI and HVII Results | 15 1 |
Bone 1 | Tooth | 22.1 | 49.3 | 13,650 | Full STR Identifiler + MiniFiler; HVI/HVII Results Outsourced | 10 2 |
Bone 2 | Foot Bone | 0.1 | 0.5 | 1935 | Full STR Identifiler + MiniFiler; HVI/HVII Results Outsourced | 6.5 1 |
Bone 3 | Femur | 0.5 | 1.1 | 6550 | No STR; HVI/HVII Results Outsourced | 6.5 1 |
Bone 4 | Skull | 0.1 | 0.2 | 670 | No STR; Partial HVI/HVII Results | 8 1 |
Bone 5 | Femur | 0 | 0 | N/A | No STR; No HVI/HVII Results | 7.2 1 |
Bone 6 | Femur | 0 | N/A | 1890 | 3 STRs Identifiler + MiniFiler; No HVI/HVII Results | 13.2 1 |
Bone 7 | Pelvic Bone | 0 | 7200 | N/A | No STR; No HVI/HVII Results | 10 2 |
Bone 8 | Tooth | 0 | 0 | 3.34 | No STR; No HVI/HVII Results | 10 2 |
Bone 9 | Tooth | 0 | 48 | N/A | No STR; HVI/HVII Results Outsourced | 10 2 |
Bone 10 | Femur | 2.5 | 5.3 | N/A | Partial STR; HVI/HVII Results | 15 1 |
Bone 11 | Femur | 71 | 30,000 | N/A | Partial STR; No HVI/HVII Results | 5 4 |
Bone 12 | Tooth | 4.6 | 71.4 | N/A | Partial STR; No HVI/HVII Results | 5 4 |
Bone 13 | Tooth | 0 | 170 | N/A | Partial STR; No HVI/HVII Results | 3 4 |
Bone 14 | Femur | 88.1 | 132 | 28,000 | Partial STR; No HVI/HVII Results | 1 3 |
Tissue 1 | Tissue | 0 | 231 | 895,000 | No STR; Partial mtDNA Results | 10 2 |
Tissue 2 | Tissue | 0.1 | 658 | N/A | Partial MiniFiler; No HVI/HVII Results | 1 4 |
Tissue 3 | Tissue | N/A | N/A | N/A | Full STR with MiniFiler; Partial HVI/HVII Results | 15 1 |
Nail 1 | Nail | 370 | N/A | 17,000,000 | Full STR; HVI/HVII Results | 1 of 1/3 Dil 3 |
Nail 2 | Nail | 8900 | 53,000 | N/A | Full STR; HVI/HVII Results | 1 of 1/90 Dil 3 |
Nail 3 | Nail | 0 | 41 | N/A | No STR; No HVI/HVII Results | 8.5 1 |
Buccal 1 | Buccal | 180 | 260 | N/A | Full STR; HVI/HVII Results | 1 of 1/2 Dil 3 |
Buccal 2 | Buccal | 460 | 560 | N/A | Full STR; HVI/HVII Results | 1 of 1/4 Dil 3 |
Buccal 3 | Buccal | 320 | 360 | N/A | Full STR; HVI/HVII Results | 1 of 1/3 Dil 3 |
Converge v2.1 Analysis Setting | 8 Sample Multiplex | 16 Sample Multiplex |
---|---|---|
Minimum Total Read Coverage per Base Position | 500 reads | 250 reads |
Minimum Variants Coverage to Call | 50 reads | 25 reads |
Coverage Threshold to Mark Region | 500 reads | 250 reads |
Minimum Coverage Percent | 5% | 5% |
Confirmed Call Threshold | 90% | 90% |
Point Heteroplasmy Call or Mixed Base Threshold Call Threshold | 10% | 10% |
Insertion Call Threshold | 20% | 20% |
Deletion Call Threshold | 30% | 30% |
Run Number | Chip Loading (%) | % Polyclonal | % Low Quality Reads | Usable Reads |
---|---|---|---|---|
Run 1 | 77 | 38 | 26 | 12,174,997 |
Run 2 | 87 | 42 | 19 | 14,961,333 |
Run 3 | 84 | 37 | 52 | 8,050,849 |
Run 4 | 90 | 43 | 57 | 8,135,888 |
Run 5 | 84 | 45 | 47 | 9,107,278 |
Run 6 | 83 | 47 | 28 | 11,581,237 |
Run 7 | 87 | 54 | 33 | 9,079,330 |
Run 8 | 83 | 55 | 33 | 7,992,750 |
Run 9 | 88 | 51 | 27 | 11,247,818 |
Run 10 | 89 | 46 | 38 | 10,473,906 |
Run 11 | 89 | 49 | 27 | 11,248,640 |
Run 12 | 91 | 55 | 29 | 10,002,927 |
Sample Name | Full mtDNA Coverage | Sanger CA DOJ | Sanger CA DOJ and Outsourcing Combined Results | Precision ID Assay |
---|---|---|---|---|
Hair 1–1 | 100% | ☐ | ☐ | ☒ |
Hair 1–2 | 93% | ☐ | ☐ | ☐ |
Hair 1–3 | 75% | ☐ | ☐ | ☐ |
Hair 2–1 | 95% | ☐ | ☐ | ☒ |
Hair 2–2 | 93% | ☐ | ☐ | ☐ |
Hair 3–1 | 96% | ☐ | ☐ | ☐ |
Hair 3–2 | 93% | ☐ | ☐ | ☒ |
Hair 4 | 99.4% | ☒ | ☒ | ☐ |
Bone 1 | 99.5% | ☒ | ☒ | |
Bone 2 | 99.4% | ☒ | ☒ | |
Bone 3 | 100% | ☐ | ☒ | ☒ |
Bone 4 | 98.8% | ☐ | ☐ | ☒ |
Bone 5 | 52% * | ☐ | ☐ | ☐ * |
Bone 6 | 96.7% | ☐ | ☐ | ☒ |
Bone 7 | 84% | ☐ | ☐ | ☒ |
Bone 8 | 0% | ☐ | ☐ | ☐ |
Bone 9 | 0% | ☐ | ☐ | |
Bone 10 | 85% | ☒ | ☒ | ☒ |
Bone 11 | 100% | ☐ | ☐ | ☒ |
Bone 12 | 100% | ☐ | ☐ | ☒ |
Bone 13 | 99.5% | ☐ | ☐ | ☒ |
Bone 14 | 100% | ☐ | ☐ | ☒ |
Tissue 1 | 91.3% | ☐ | ☐ | ☒ |
Tissue 2 | 100% | ☐ | ☐ | ☒ |
Tissue 3 | 100% | ☐ | ☐ | ☒ |
Nail 1 | 100% | ☒ | ☒ | ☒ |
Nail 2 | 100% | ☒ | ☒ | ☒ |
Nail 3 | 100% | ☐ | ☐ | ☒ |
Buccal 1 | 99.2% | ☒ | ☒ | ☒ |
Buccal 2 | 100% | ☒ | ☒ | ☒ |
Buccal 3 | 85% | ☒ | ☒ | ☒ |
Success | Rate | 7/28 = 25% | 10/31 = 32.3% | 23/31 = 74.2% |
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Cuenca, D.; Battaglia, J.; Halsing, M.; Sheehan, S. Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay. Genes 2020, 11, 1303. https://doi.org/10.3390/genes11111303
Cuenca D, Battaglia J, Halsing M, Sheehan S. Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay. Genes. 2020; 11(11):1303. https://doi.org/10.3390/genes11111303
Chicago/Turabian StyleCuenca, Daniela, Jessica Battaglia, Michelle Halsing, and Sandra Sheehan. 2020. "Mitochondrial Sequencing of Missing Persons DNA Casework by Implementing Thermo Fisher’s Precision ID mtDNA Whole Genome Assay" Genes 11, no. 11: 1303. https://doi.org/10.3390/genes11111303