of NGS technology for identification, and phenotypic ... · • IGNA (Nantes Atlantic Genetic Institute) was created in 2003 by Pr.Moisan • The biggest privateforensic lab in France

Evaluation of NGS technology for identification, ancestry prediction and phenotypic characters

prediction with the Ion PGM™ System

Who we are ?

• IGNA (Nantes Atlantic Genetic Institute) wascreated in 2003 by Pr. Moisan

• The biggest private forensic lab in France

• 2 labs : Nantes and Marseille

• 55 persons (49 in Nantes and 6 in Marseille)

• IGNA is ISO 17025 accredited since 2010

• IGNA it’s more than 1 million of geneticprofiles in 12 years

Timing

• Since June 2014, SNPs analysis can be used in France for ancestry andphenotypic characters predictions from an unknown DNA in criminal cases

• May 2015, an Ion PGM™ System was installed at IGNA

• September 2015, IGNA performed its first NGS analysis for the Frenchjustice

• From May to August : what are the performances of the NGS analysis foridentification, ancestry prediction and phenotypic characters predictionfrom an unknown DNA ?

New technology, many questions

• Identification with Identity Panel– What is the sensitivity of this panel ?

– What are the performances of this panel for degraded DNA ?

– What is the discrimination power of SNPs analysis ?

• Ancestry prediction with Ancestry Panel– Is this panel reliable ?

• Phenotypic characters prediction– Can we use the Ion PGM™ System for

eye and hair colour prediction ?

Many questions that need to be answered

SNP Test Conditions

• SNPs analysis performed in Thermo Fisher Scientific conditions• DNA amplification using Identity and Ancestry panels and HIrisplex system

• Minimum criteria for the validation of SNPs

• Coverage

Autosomal DNA : QDNA/PCR amplification ≥ 50pg : coverage ≥ 100 readsQDNA/PCR amplification < 50pg : coverage ≥ 400 reads

Y marker : coverage ≥ 50 reads

• Percentage of forward and reverse strand : 30% ≤ ratio ≤ 70%

• Major Allele Frequence (number of MAF reads in total reads number) :

Homozygous alleles ≥ 95% 50% ≤ Heterozygous alleles ≤ 60%

SNP Test Conditions

• Valid alleles vs. drop out/in alleles according to the coverage (autosomal

SNPs only)

Coverage ≥ 100 reads for all DNA quantities

0

10

20

30

40

50

60

70

80

90

600 300 150 100 50 25 12,5 6,25 3,125

Num

bero

f alleles

Quantity of 007 DNA control (pg/amplification)

Validalleles

Drop out /in alleles

Coverage ≥ 100 reads for QDNA/PCR amplification ≥ 50pg

Coverage ≥ 400 reads for QDNA/PCR amplification < 50pg

0

10

20

30

40

50

60

70

80

90

600 300 150 100 50 25 12,5 6,25 3,125

Num

ber o

f alleles


Validalleles

Drop inalleles

STR Test Conditions

• STR analysis performed in Thermo Fisher Scientific conditions

• GlobalFiler PCR amplifications : 25μl and 29 PCR cycles

• Electrophoresis : 3500xL and injection conditions 1.2kV/24 sec

• Minimum criteria for the validation of alleles

• Homozygous alleles ≥ 1600 RFU

• Heterozygous alleles ≥ 200 RFU

• PHR ≥ 60%

Identity Panel : Sensitivity

• Sensitivity comparison of the STR analysis to the autosomal SNP analysis

(without hemizygous alleles)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

600 300 150 100 50 25 12.5 6.25 3.125

% of v

alid

alleles

Quantity of 007 DNA control (pg/PCR amplification)

Percentage of valid alleles

STR Analysis

SNP Analysis

1E‐401E‐371E‐341E‐311E‐281E‐251E‐221E‐191E‐161E‐131E‐10

0,00000010,0001

0,1

600 300 150 100 50 25 12.5 6.25 3.125

Freq

uencyin gen

eralpo

pulatio

n

Quantity of 007 DNA control (pg/PCR amplification)

Frequency of STR and SNP genetic profiles

STR analysis

SNP analysis


6.25pg/PCR amplification , STR 0%, SNP 19% 3.125pg/PCR amplification , STR 0%, SNP 4%

25pg/PCR amplification, STR 29%, SNP 81% 12.5pg/PCR amplification , STR 0%, SNP 38%


• Identity panel sensitivity with and without hemizygous alleles

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

600 300 150 100 50 25 12.5 6.25 3.125

% of v

alid

alleles


Percentage of valid alleles

Withouthemizygousalleles

Withhemizygousalleles

1E‐40

1E‐37

1E‐34

1E‐31

1E‐28

1E‐25

1E‐22

1E‐19

1E‐16

1E‐13

1E‐10

0,0000001

0,0001

0,1

600 300 150 100 50 25 12.5 6.25 3.125

Freq

uencyin gen

eralpo

pulatio

n


Frequency of SNPs genetic profiles

Withouthemizygousalleles

Withhemizygousalleles

Identity Panel for Degraded DNA

• 4 casework samples analysed in NGS and in STR

Degradation Index : 512Degradation Index : 30

Degradation Index : 12Degradation Index : 5

Identity Panel for Degraded DNA

0

100

200

300

400

500

600

700

800

0%10%20%30%40%50%60%70%80%90%

100%

pg

% of v

alid

alleles

Casework sample

Percentage of valid alleles according to the DNA quantity per PCR amplification

STR analysis

SNP analysis (withouthemizygous alleles)

SNP analysis (withhemizygous alleles)

DNA quantity/PCR

DI : 5 12 30 512

0

100

200

300

400

500

600

0%10%20%30%40%50%60%70%80%90%100%

Degrada

tion inde

x

% of v

alid

alleles

Casework sample

Percentage of valid alleles according to the degradation index (DI)

STR analysis



Degradation index

DI : 5 12 30 512

01002003004005006007008001E‐34

1E‐311E‐281E‐251E‐221E‐191E‐161E‐131E‐10

0,00000010,0001

0,1

pg

Freq

ency

Casework sample

Frequency of genetic profiles according tothe DNA quantity per PCR amplification

STR analysis



DNA quantity/PCR

0

100

200

300

400

500

6001E‐341E‐311E‐281E‐251E‐221E‐191E‐161E‐131E‐10

0,00000010,0001

0,1

Degrada

tion inde

x

Freq

ency

Casework sample

Frequency of genetic profiles according tothe degradation index (DI)

STR analysis



Degradation index

Ancestry Prediction

• 4 persons from different origins

Admixture prediction

Population likelihoods

Ancestry Prediction

• A family with parents from different origins and their child

Eye and Hair Colour Prediction

• Simultaneous prediction of hair and eye colour from DNA with the HIrisplex

system


Eye Colour Predicted Probability

Blue Eye 0.21

Intermediate Eye 0.16

Brown Eye 0.63


Blue Eye 0.00


Brown Eye 0.97

Person 1DNA analysis



Blue Eye 0.03


Brown Eye 0.88


Predicted PredictedPredicted

Observed ObservedObserved





Blue Eye 0.90


Brown Eye 0.04


Blue Eye 0.00


Brown Eye 0.99

Predicted

Observed

Predicted

Observed


Eye Colour PredictedProbability

Blue Eye 0,03

Intermediate Eye 0,09

Brown Eye 0,88

Eye Colour PredictedProbability

Blue Eye 0,04

Intermediate Eye 0,10

Brown Eye 0,86


Predicted Predicted


Observed


Person ADNA analysis

Person BDNA analysis

Hair Shade Predicted Probability

Light Hair 0.009Dark Hair 0.991

Hair Colour Predicted Probability

Brown Hair 0.263Red Hair 0.000Black Hair 0.731Blond Hair 0.006


Light Hair 0.890

Dark Hair 0.110


Brown Hair 0.112

Red Hair 0.873

Black Hair 0.001

Blond Hair 0.014

Person CDNA analysis





Predicted Predicted Predicted

Observed Observed Observed


Person DDNA analysis





Predicted

Observed

Person EDNA analysis





Predicted

Observed

Summary

• NGS analysis with Identity panel– More sensitive than STR analysis on CE

– Much more efficient for degraded DNA analysis than STR analysis on CE

– Higher discriminating power than STR analysis

– Unfortunately, forensic DNA database with SNPs genetic profiles doesn’t exist inFrance and anywhere else in the world

• NGS analysis with Ancestry panel– Good concordance between DNA analysis prediction and people origin

• NGS analysis with HIrisplex system– Good concordance between DNA analysis prediction and hair colour of people

– Quite good concordance between DNA analysis prediction and eye colour of people

Conclusion

NGS analysis using Ion PGM™ System from Thermo Fisher Scientific is

a new and performant approach in genetic forensic analysis for

identification, ancestry prediction and phenotypic characters prediction

Thank you for your attention !

When used for purposes other than Human Identification the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures.

Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration

Documents

of NGS technology for identification, and phenotypic ... · • IGNA (Nantes Atlantic Genetic Institute) was created in 2003 by Pr.Moisan • The biggest privateforensic lab in France