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Validation and implementation of the Precision ID mtDNA Whole Genome Panel kit for use in paternity and immigration cases

Anders Buchard

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Section of Forensic Genetics

Department of Forensic Medicine

Faculty of Health and Medical Sciences

University of Copenhagen

Denmark

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Agenda

  • Introduction to the Precision ID mtDNA kit

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  • Validation data

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  • Super short presentation of the results

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  • A run through of the analysis parameters and restrictions we have chosen to impose on the data in order to be confident in our ability to create reproducible genotypes

UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Amplification done in two separate multiplexes

81 primer pairs in each multiplex

UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Simplified workflow

2-separate multiplex PCR

Library Build (pooled PCRs)

Emulsion PCR (Ion-chef)

Sequencing (PGM/S5)

Data analysing

UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Validation data

Samples:

  • 95 Danes
  • 2 x PGM og 2 x S5

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Analysis:

  • Variantcaller plugin and Excel macro
  • Converge and Excel macro

UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Results (super short)

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  • PGM vs PGM (Variantcaller): Inconsistencies and surprises

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  • PGM vs S5 (Variantcaller): Inconsistencies and new surprises

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  • S5 Variantcaller vs S5 Converge: Inconsistencies and new surprises

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  • S5 Converge vs S5 Converge: Full profile match when using our analysis/reporting rules

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

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  • Minimum sequencing depth: 100 reads

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  • Noise filter: 7%

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  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

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  • Strand bias: Positions with a strand bias of more than 0.8

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  • Deletions/insertions: Not reported

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  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

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  • Minimum sequencing depth: 100 reads

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  • Noise filter: 7%

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  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

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  • Strand bias: Positions with a strand bias of more than 0.8

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  • Deletions/insertions: Not reported

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  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

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  • Minimum sequencing depth: 100 reads

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  • Noise filter: 7%

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  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

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  • Strand bias: Positions with a strand bias of more than 0.8

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  • Deletions/insertions: Not reported

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  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

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  • Minimum sequencing depth: 100 reads

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  • Noise filter: 7%

​

  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

​

  • Strand bias: Positions with a strand bias of more than 0.8

​

  • Deletions/insertions: Not reported

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  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

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  • Minimum sequencing depth: 100 reads

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  • Noise filter: 7%

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  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

​

  • Strand bias: Positions with a strand bias of more than 0.8

​

  • Deletions/insertions: Not reported

​

  • Positions not reported: Positions between 301-316 and 16179- 16193

​

  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

​

  • Minimum sequencing depth: 100 reads

​

  • Noise filter: 7%

​

  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

​

  • Strand bias: Positions with a strand bias of more than 0.8

​

  • Deletions/insertions: Not reported

​

  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Analysis rules (imposed on data from Converge in Excel)

​

  • Minimum sequencing depth: 100 reads

​

  • Noise filter: 7%

​

  • Heteroplasmy: Variants with frequencies between 15% and 85% (not reported)

​

  • Strand bias: Positions with a strand bias of more than 0.8

​

  • Deletions/insertions: Not reported

​

  • Positions not reported: Positions between 301-316 and 16179- 16193

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  • Warnings in Converge for ”NUMTS” and ”Degraded”

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE

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Final remarks

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  • Despite the conservative approach we end up with 93 different haplotypes from 95 samples

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  • It lies in the nature of MPS that a little tweak in the pipeline, e.g. alignment version, will have an effect on how the results are being reported. We will not be able to avoid that except if we use stable, never-changing settings

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  • Meta data: We will register analysis software (version), sequencing platform and imposed analysis rules for each profile

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UNIVERSITY OF COPENHAGEN FACULTY OF HEALTH AND MEDICAL SCIENCES DEPARTMENT OF FORENSIC MEDICINE