Evaluation of NGS technology for identification, ancestry prediction and phenotypic characters prediction with the Ion PGM System
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1 Evaluation of NGS technology for identification, ancestry prediction and phenotypic characters prediction with the Ion PGM System
2 Who we are? IGNA (Nantes Atlantic Genetic Institute) was created 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 accredited since 2010 IGNA it s more than 1 million of genetic profiles in 12 years
3 Timing Since June 2014, SNPs analysis can be used in France for ancestry and phenotypic 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 French justice From May to August : what are the performances of the NGS analysis for identification, ancestry prediction and phenotypic characters prediction from an unknown DNA?
4 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
5 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 reads QDNA/PCR amplification < 50pg :coverage 400 reads Ymarker: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%
6 SNP Test Conditions Valid alleles vs. drop out/in alleles according to the coverage (autosomal SNPs only) Coverage 100 reads for all DNA quantities Coverage 100 reads for QDNA/PCR amplification 50pg Coverage 400 reads for QDNA/PCR amplification < 50pg Number of alleles Valid alleles Drop out / in alleles Number of alleles Valid alleles Drop in alleles ,5 6,25 3, ,5 6,25 3,125 Quantity of 007 DNA control (pg/amplification) Quantity of 007 DNA control (pg/amplification)
7 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%
8 Identity Panel : Sensitivity Sensitivity comparison of the STR analysis to the autosomal SNP analysis (without hemizygous alleles) Percentage of valid alleles Frequency of STR and SNP genetic profiles % of valid alleles 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% STR Analysis SNP Analysis 1E 40 1E 37 1E 34 1E 31 1E 28 1E 25 1E 22 1E 19 1E 16 1E 13 1E 10 0, ,0001 0,1 Frequency in general population STR analysis SNP analysis Quantity of 007 DNA control (pg/pcr amplification) Quantity of 007 DNA control (pg/pcr amplification)
9 Identity Panel : Sensitivity 25pg/PCR amplification, STR 29%, SNP 81% 12.5pg/PCR amplification, STR 0%, SNP 38% 6.25pg/PCR amplification, STR 0%, SNP 19% 3.125pg/PCR amplification, STR 0%, SNP 4%
10 Identity Panel : Sensitivity Identity panel sensitivity with and without hemizygous alleles Percentage of valid alleles Frequency of SNPs genetic profiles % of valid alleles 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Without hemizygous alleles With hemizygous alleles Frequency in general population 1E 40 1E 37 1E 34 1E 31 1E 28 1E 25 1E 22 1E 19 1E 16 1E 13 1E 10 0, ,0001 0, Without hemizygous alleles With hemizygous alleles Quantity of 007 DNA control (pg/amplification) Quantity of 007 DNA control (pg/amplification)
11 Identity Panel for Degraded DNA 4 casework samples analysed in NGS and in STR Degradation Index : 5 Degradation Index : 12 Degradation Index : 30 Degradation Index : 512
12 Identity Panel for Degraded DNA Percentage of valid alleles according to the degradation index (DI) Percentage of valid alleles according to the DNA quantity per PCR amplification % of valid alleles 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Degradation index STR analysis SNP analysis (without hemizygous alleles) SNP analysis (with hemizygous alleles) Degradation index % of valid alleles 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% pg STR analysis SNP analysis (without hemizygous alleles) SNP analysis (with hemizygous alleles) DNA quantity/pcr Casework sample DI : Casework sample DI : Frequency of genetic profiles according to the degradation index (DI) Frequency of genetic profiles according to the DNA quantity per PCR amplification 1E 34 1E 31 1E 28 1E 25 1E 22 1E 19 1E 16 1E 13 1E 10 0, ,0001 0,1 Freqency Degradation index STR analysis SNP analysis (without hemizygous alleles) SNP analysis (with hemizygous alleles) Degradation index 1E 34 1E 31 1E 28 1E 25 1E 22 1E 19 1E 16 1E 13 1E 10 0, ,0001 0,1 Freqency pg STR analysis SNP analysis (without hemizygous alleles) SNP analysis (with hemizygous alleles) DNA quantity/pcr Casework sample Casework sample
13 Ancestry Prediction 4 persons from different origins Population likelihoods Admixture prediction
14 Ancestry Prediction A family with parents from different origins and their child
15 Eye and Hair Colour Prediction Simultaneous prediction of hair and eye colour from DNA with the HIrisplex system
16 Eye and Hair Colour Prediction Person 1 Person 2 Person 3 Eye Colour Predicted Probability Eye Colour Predicted Probability Eye Colour Predicted Probability Blue Eye 0.21 Intermediate Eye 0.16 Brown Eye 0.63 Blue Eye 0.03 Intermediate Eye 0.09 Brown Eye 0.88 Blue Eye 0.00 Intermediate Eye 0.03 Brown Eye 0.97 Predicted Predicted Predicted Observed Observed Observed
17 Eye and Hair Colour Prediction Person 4 Person 5 Eye Colour Predicted Probability Eye Colour Predicted Probability Blue Eye 0.90 Intermediate Eye 0.07 Brown Eye 0.04 Predicted Blue Eye 0.00 Intermediate Eye 0.01 Brown Eye 0.99 Predicted Observed Observed
18 Eye and Hair Colour Prediction Person 2 Person 6 Eye Colour Predicted Probability Blue Eye 0,03 Predicted Predicted Eye Colour Predicted Probability Blue Eye 0,04 Intermediate Eye 0,09 Intermediate Eye 0,10 Brown Eye 0,88 Brown Eye 0,86 Observed
19 Eye and Hair Colour Prediction Person A Hair Shade Predicted Probability Light Hair Dark Hair Person B Hair Shade Predicted Probability Light Hair Dark Hair Person C Hair Shade Predicted Probability Light Hair Dark Hair Hair Colour Predicted Probability Brown Hair Red Hair Black Hair Blond Hair Hair Colour Predicted Probability Brown Hair Red Hair Black Hair Blond Hair Hair Colour Predicted Probability Brown Hair Red Hair Black Hair Blond Hair Predicted Predicted Predicted Observed Observed Observed
20 Eye and Hair Colour Prediction Person D Hair Shade Predicted Probability Light Hair Dark Hair Person E Hair Shade Predicted Probability Light Hair Dark Hair Hair Colour Predicted Probability Brown Hair Red Hair Black Hair Blond Hair Predicted Hair Colour Predicted Probability Brown Hair Red Hair Black Hair Blond Hair Predicted Observed Observed
21 Summary NGS analysis with Identity panel More sensitive than STR analysis on CE Much more efficient for degraded than STR analysis on CE Higher discriminating power than STR analysis Unfortunately, forensic DNA database with SNPs genetic profiles doesn t exist in France and anywhere else in the world NGS analysis with Ancestry panel Good concordance between prediction and people origin NGS analysis with HIrisplex system Good concordance between prediction and hair colour of people Quite good concordance between prediction and eye colour of people
22 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
23 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
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