The world leader in serving science

Gottfried WeichholdSr Application Specialist HID Europe

Seeking Answers from Challenging Cases with NGS

2

• What types of cases cannot easily be solved with traditional DNA analysis methods?

• What are SWGDAM validation guidelines and implementation challenges for an NGS system?

• What are legislative considerations surrounding NGS implementation?

Main Topics

3

~70% of cases likely give CE STR results. What about the 30%?

4

NGS is a key to getting more information for forensics

Next-generation Sequencing

Sequencing allows greater depth of genetic information

Multiplexing hundreds of the same types of markers

i.e. SNPs

Multiplex different types of markers i.e. SNP+ STR

Simultaneously analyze multiple samples (barcoding)

Target small amplicons to recover DNA from challenging

samples

5

When current CE-based methods can’t resolve a case…

…NGS can supplement traditional CE STR testing withforensically relevant, targeted panels for specific uses.

• Low quality and low quantity samples (mtDNA)• Precision ID mtDNA Whole Genome Panel• Precision ID mtDNA Control Region Panel

• Degraded samples (identity SNPs)• Precision ID Identity Panel

• Investigative leads (ancestry and phenotypic SNPs)• Precision ID Ancestry Panel• Ion AmpliSeq™ DNA Phenotyping Panel

• Complex Mixtures (NGS STRs)• Precision ID GlobalFiler™ NGS STR Panel v2

6

• Total hands-on time: less than 45 minutes • Total overall time: as little as 25 hours• Converge Software analysis modules for mtDNA, SNPs and STRs

• HPS validation services now available for Precision ID NGS System

Simplest targeted NGS workflow

7

Developed for Highly Compromised and Degraded SamplesPrecision ID mtDNA Panel Specification

8

Sensitivity Titration Experiment – mtGenome

• Average mtDNA copies/cell w/ 0.1ng gDNA = 2,900• mtDNA copies will vary among cell types.

* (a-d) 100, 10, 5 and 2 pg gDNA input* 2 DNA sources

Coverage is consistent for all template inputs; would need to test below 2pg to see decrease.

9

Case Study- mtDNA from blood stains

• Samples ranged in age from 2 months to 12 years

• Stored on gauze, swabs, and FTA at room temperature

• Concordance with Sanger – 99.86%

11

• Remains from Japanese cemetery could be 300 years old

• Previous study indicated 36% of Japanese typed as haplogroup D4

• This study showed all 3 remains as D4a1C

• Control Region typing only would not have discriminated between the individuals

• Only the Whole Genome typing could accomplish this haplogroup determination

Case study- aged remains

12

• Control region sequencing from isolated indigenous populations

• Ayoreo ethnic group (western Paraguay): 80% haplogroup C and 20% haplogroup D

• Ache ethnic group (eastern Paraguay): 90% were from haplogroup B and 10% from haplogroup A

• Whole genome sequencing from urban Alto Parana

• 84 unique haplotypes

• 9 shared by 2 individuals

• 1 shared by 3 individuals

• WG comparisons to CR showed different haplogroup in 26%

Case study- maternal lineage analysis

13

Case Study- unidentified remains

• mtDNA – maternal lineage• YSTR – paternal lineage• Phenotypic SNPs – physical traits• Ancestry SNPs – biogeographic ancestry

15

• Distinguish stutter from true alleles• Mixture Sample consisting of:

• Major: 11,14• Minor: 13,14

Case study - detecting multiple contributors

16

Implementation Challenges

• Validation• FBI’s Quality Assurance Standards / Interpretation Criteria• NDIS Approval• Future considerations

• Advanced Statistics / Bioinformatics • Integration w/ Probabilistic Genotyping

17

• SWGDAM Validation Guidelines for DNA Analysis Methods issued Dec 2016

• Specific NGS recommendations• Effects of barcoding

• Limit of detection on input DNA and quality/quantity of libraries pooled

• Crosstalk during sequencing and carryover between runs

• Precision ID mtDNA Panels validation in collaboration with KOLs in 2019

• Precision ID GlobalFiler NGS STR v2 Panel validation in collaboration with KOL’s in 2019

What does SWGDAM say about using NGS?

18

New SWGDAM interpretation guidelines published (April 2019)

Interpretation guidelines enable NDIS approval

19

The Precision ID mtDNA solution is NDIS approved

The U.S. Federal Bureau of Investigation (FBI) has approved the Applied Biosystems™ Precision ID mtDNAsolution for use by forensic laboratories to generate mitochondrial DNA (mtDNA) profiles for uploading to the U.S. National DNA Index System (NDIS) database.

20

Published Validation Studies

21

Why has forensic community been slow to adopt NGS STRs?

• Not validated for casework

• No match statistics/ population frequencies for new STR alleles

• NIST publication as well as others are generating these needed frequencies

• Not accepted by courts yet• will need admissibility hearings

22

Legal & Database Considerations – International Perspective

• Genetic Privacy Concerns• Varies by country

• National Databases• Data acceptance

• New information from STRs− Nomenclature adoption

• New marker adoption

23

Ion AmpliSeq Identity Panel, Ion AmpliSeq Ancestry Panel, Precision ID GlobalFiler NGS STR Panel v2, Precision ID Ancestry Panel, Precision ID Identity Panel, Precision ID mtDNA Whole Genome Panel and Precision ID mtDNA Control Region Panel are For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. When used for purposes other than Human Identification, the instrument and the modules of the cited software are for Research Use Only. Not for use in diagnostic procedures

For licensing and limited use restrictions visit HIDlicensing.

©2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

thermofisher.com/HID-NGS

Thank you for your attention!Questions?

THANK YOU!