development of a next generation y-str multiplex for forensic applications

1 Life Technologies™ |

Development of a Next Generation Y-STR Multiplex for Forensic Applications 2013 Future Trends in Forensic DNA Technology Seminar Series August 15, 2013


Agenda

§  Introduction to Y-STR Usage in Forensic Science

§  Development Criteria for a new Next Generation Y-STR (NGY) Multiplex

§  NGY Kit design and configuration

§  Internal/External Test Site Data

§  Preliminary work on discrimination of close relatives with the NGY kit


Forensic Applications of Y-STR Analysis

§  Y-STR analysis is a valuable tool in certain forensic cases: −  Detection of male DNA when mixed with an excess of female

DNA >  Sexual assault cases >  Useful for exclusion

−  Dealing with multiple male contributors −  Tracing family relationship among males

>  Patrilineal inheritance

−  Missing person investigations −  Paternity testing

>  Alleged father not available

§  Haplotype databases are used to establish the frequency of occurrence of a haplotype in specific populations


AmpFSTR® Yfiler® Kits

Proven to deliver excellent results with casework samples

Designed for high-quality results with minimal steps for single source

reference samples

AmpFSTR® Yfiler® Kit

AmpFSTR® Yfiler® Direct Kit


What is the single most important factor for you in selecting a new Y-STR kit?

1.  Discrimination Power 2.  Performance with Challenging Samples 3.  Time to Result/Ease of Use 4.  Size of Haplotype Databases for Frequency Estimations 5.  No plans to evaluate a new Y-STR kit


Limitations of Current Yfiler® Kits

Y-STR Set

Performance with Challenging samples

Workflow & Data Analysis

§  Haplotype resolution varies across populations1

§  Male relatives of a suspect cannot be excluded2

§  Adventitious matches increase as more male profiles are added to Y-STR frequency databases

§  Lower sensitivity and ability to overcome inhibition as compared to autosomal STR kits

§  Limited performance with low-ratio male/female mixture samples

§  Longer time to results as compared to newer generation autosomal STR kits

§  Allelic ladder not up to date with new reported alleles

1Vermeulen et al. (2009): FSIG 3: 205-213 2Ballantyne et al. (2010): Am J Hum Genet 87:341-353


Development Criteria

Maintain concordance with existing Y-STR databases

by keeping AmpFℓSTR® Yfiler® markers

Improve discrimination capacity by including new Y-STR markers

Improve inhibitor resistance and sensitivity by leveraging advancements of next generation STR-Kits

Improve Performance of existing AmpFℓSTR®

Yfiler® markers


Concordance with Existing Y-STR Data

§  Y-STR Haplotype Statistics −  YHRD: over 110,000 haplotypes −  US Y STR Database: ~ 25,000

haplotypes −  Yfiler Database: over 11,000

haplotypes

§  More than 50,000 Yfiler® haplotypes in Y-STR frequency databases

§  Replacement of existing profiles would not be feasible


Mutation Rate

Measures the mutation rate per locus per generation

Key Parameters for Y-STR Markers

Gene Diversity (GD)

Discrimination Capacity (DC)

Measures the uniqueness of a particular marker in a given population

Measures the number of unique haplotypes in a given population

Population of N Marker Y

1 Haplotype GD = 0

DC = 1/N

N Haplotypes GD = 0.99

DC = 1

Standard Y-STR Markers à 1 × 10-3 and 1 × 10-4 per locus per generation

Rapidly Mutating (RM) Y-STR Markers à 1 × 10-2 per locus per generation

Adapted from M. D. Coble, Examination of Rapidly Mutating Y-STR Loci Increased Resolution of Common Haplotypes


Presence of Differentiating Mutations in Male Relatives

Yfiler® Kit

RM Y-STRs

Number of Meioses Separating Pair Pe

rcen

tage

of R

elat

ive

Pairs

Diff

eren

tiate

d by

1+

Mut

atio

n

K.N. Ballantyne , Forensic Science Review, 2012

156 pairs separated by 1-20 generation assessed with Yfiler and RM Y-STR set (13 loci)


Selection Criteria for New Y-STR Markers

High gene diversity Minimize adventitious matches

Markers with higher mutation rate Distinguish between close relatives

Markers with lower mutation rate Improve lineage differentiation

Maximum 2 copies per marker Simplify data analysis

Increase number of mini-STRs Facilitate analysis of degraded DNA

Primer compatibility Reduce risk of artifacts

Male specificity Increase data accuracy


Selection Criteria for Yfiler® Plus Markers: Gene Diversity vs. Mutation Rate of Y-STR

markers

0.0E+00

5.0E-‐03

1.0E-‐02

1.5E-‐02

2.0E-‐02

2.5E-‐02

0.0 0.2 0.4 0.6 0.8 1.0

Mutation Ra

te

Gene Diversity

Yfiler® kit STRsOther Y-‐STRs

Mutation Rate: Ballantyne K et al. (2010): Am J Hum Genet 87: 341-353 Gene Diversity: various publications


Selection Criteria for Yfiler® Plus Markers: Gene Diversity vs. Mutation Rate of Y-STR

markers

Mutation Rate: Ballantyne K et al. (2010): Am J Hum Genet 87: 341-353 Gene Diversity: various publications

0.0E+00

5.0E-‐03

1.0E-‐02

1.5E-‐02

2.0E-‐02

2.5E-‐02

0.0 0.2 0.4 0.6 0.8 1.0

Mutation Ra

te

Gene Diversity

Yfiler® kit STRsnew NGY STRsOther Y-‐STRsYfiler® Plus new STRs


Fall 2013

Yfiler® Plus: The First 6-Dye, 27 Marker Y-STR Multiplex

Plus in Power of Discrimination

•  All 17 loci amplified by the Yfiler® kit •  10 new Y-STR loci including 7 Rapidly Mutating1 Y-STRs for ability to

discriminate between close male relatives •  11 mini STRs below 220bp

Plus in Performance

•  Improved sensitivity & results with challenging casework samples •  Improved recovery of alleles and intracolor balance in male/female

mixtures containing high female background •  Improved resolution capacity for male/male mixtures •  Expanded allelic ladder •  Shorter amplification times •  Direct amplification protocol with single-source samples

1.  Rapidly Mutating Y-STRs have mutation rates above 1 x 10-2 per marker per generation. 17 Yfiler Y-STRs show mutation rates between 1 x 10-3 and 1 x 10-4 per marker per generation. The content provided herein may relate to products that have not been officially released and is subject to change without notice.


50 100 150 200 250 300 350 400

DYS627DYS389IIDYS635DYS389IDYS576

DYS391DYS448

Y GATA H4DYS19DYS458DYS460

DYS518DYS392DYS438DYS390DYS456

DYS449DYS385ab

DYS437DYS570

DYS533DYF387S1ab

DYS481DYS439DYS393

Yfiler® Plus Kit Configuration

Rapidly Mutating Y-STR Standard Y-STR New Y-STR Markers →

1

3

6

8

10

2

4 5

7

9

11


Data courtesy of Mike D. Coble, Ph.D.

Discrimination Capacity of the Yfiler® Plus Kit

*Based on theoretical calculations performed by NIST – Applied Genetics

Group

** DYS460 was not included in the calculations


Yfiler® Plus Thermal Cycling Protocols

PCR Protocol

95°C/ 1 min 94°C/ 4 sec 61°C/ 60 sec 60°C/ 22 min

30x

Extracted DNA for Casework

Direct Amp for Single-Source

Samples

PCR Protocol

95°C/ 1 min 94°C/ 4 sec 61°C/ 60 sec 60°C/ 22 min

25-29x

Two Workflows

One Kit

Amp Time ~ 92 min Amp Time ~ 90 min


Yfiler® Plus Kit Development 007 Control DNA Profile

1ng of male 007 control DNA amplified for 30 cycles


Yfiler® Plus Kit Development Direct Amplification of Blood on Treated Paper

1.2 mm FTA® disc amplified for 27 cycles


Yfiler® Plus Kit Development Direct Amplification of Buccal on Treated Paper

1.2 mm FTA® disc amplified for 28 cycles


Yfiler® Plus Allelic Ladder

Alleles Virtual Bins Yfiler® Kits 137 72 Yfiler® Plus Kit 345 179


Allelic Ladder Improvement

§  Included new alleles reported in haplotype databases (YHRD.org, usystrdatabase.com) and other commercially available Y-STR kits

§  Yfiler® Plus multiplex includes new alleles and virtual bins:

Yfiler® Kit DYS635 range: 20-26 Virtual bins: 19

Yfiler® Plus Kit DYS635 range: 15-30 Virtual bins: 14, 21.3, 31


Yfiler® Plus Kit Development Instrument Compatibility

3500xL 3130xl*

*6-Dye Compatibility with Data Collection Software v4 & 6-Dye module


Yfiler® Plus vs. PowerPlex® Y23

Yfiler® Plus

•  6-dye kit •  27 Y-STR Loci •  7 Rapidly Mutating Y-STR loci •  Includes all 17 Yfiler® loci •  Includes all the PowerPlex® Y23

loci with the exception of DYS549 and DYS643

•  11 loci below 220 bp (mini-STR) •  Amplification times: casework

samples: 92 min, single source samples: 90 min (direct amp)

PowerPlex® Y23

•  5 dye kit •  23 Y-STR Loci •  2 Rapidly Mutating Y-STR loci •  Includes all 17 Yfiler® loci

•  8 loci below 220 bp (mini-STR) •  Amplification times: casework

samples: 100 min, single source samples: 90 min (direct amp)


Validation Studies

Summary of Results


Internal VTS Data


Casework Protocol

§  Study Plan: −  Experiment 1: Sensitivity Study

>  007 Control DNA Dilution Series:1 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.25 pg & NTC in replicates of 4

−  Experiment 2: Inhibited DNA Study >  1 ng 007 Control DNA + 20 µM & 200 µM Hematin >  1 ng 007 Control DNA + 10 ng/µL & 100 ng/µL Humic Acid >  All done in replicates of 4

−  Experiment 3: Mixture Study >  Two Male:Female mixture ratios: 1:1000 & 1:4000 in replicates of 3

§  Experiments 1-3 to be performed with the Yfiler® Plus Kit, Yfiler® Kit, and PowerPlex® Y23.


Casework Protocol - Sensitivity Study

Greater number of alleles recovered by the Yfiler® Plus multiplex compared to the Yfiler® Kit Data from internal validation

32pg62pg125pg250pg500pg1ng

25

20

15

10

Alle

le C

ount

17

23

27 Yfiler PlusPPY23Yfiler

Kit

12.75

9

14

15.5

17.5

25.25

17

22.75

27

15.75

23

27

17

23

27

17

23

27

Sensitivity: Alleles recovered


Casework Protocol - Male:Female Mixture Study

Yfiler® Plus multiplex shows better information recovery with male/female mixtures compared to the Yfiler® Kit Data from internal validation ; 1:1000 = 1ng M007 & 1µg F9947; 1:4000 = 0.25ng M007 & 1µg F9947

1:40001:1000

27.5

25.0

22.5

20.0

17.5

15.0

Alle

leCo

unt

17

23


Kit

16.3333

23

27

17

23

27

Mixture Study: Alleles recovered


Casework Protocol - Male:Female Mixture Study

Yfiler® Plus multiplex shows better intracolor balance with male/female mixtures compared to the Yfiler® Kit Data from internal validation ; 1:1000 = 1ng M007 & 1µg F9947; 1:4000 = 0.25ng M007 & 1µg F9947

KitSample

YfilerPPY23Yfiler Plus1:40001:10001:40001:10001:40001:1000

1.0

0.8

0.6

0.4

0.2

0.0

ICB

BGYRP

Dye

Mixture Study: ICB


Casework Protocol – Inhibition Study

Yfiler® Plus multiplex shows significant improvement with inhibitor tolerance compared to the Yfiler® Kit Data from internal validation

Humic Acid

Hemati

n 200

uM

Hemati

n 20

uM

Humic

Acid 10

0ng/

u l

Humic

Acid 10

ng/u

l

30

25

20

15

10

5

0

Alle

leCo

unt

17

23


Kit

0

23

27

15.75

23

27

0

23

27

16

23

27

Inhibition Study: Alleles recovered

27


Casework Protocol – Concordance Study

§  Data collected showed full concordance between Yfiler® and Yfiler® Plus kit

§  Exceptions were a few alleles labeled as “Off Ladder” (OL) or “Outside of Marker Range” (OMR) in Yfiler®

Yfiler® Kit

Yfiler® Plus Kit

DYS458 OL in Yfiler® Kit DYS635 OMR in Yfiler® Kit


Casework Protocol – Concordance Study

DYS456 unlabeled OMR in Yfiler® Kit

DYS456 called from a virtual bin in Yfiler® Plus STS Kit

DYS456 was expanded further and 11 is an allele in Yfiler® Plus VTS Kit


Single-Source Protocol – Performance Study

High 1st pass success rate with samples processed with direct amplification Data from three external and two internal test sites

Sample Type Test Site Cycle #

Amplifica6on First pass

success rate (100Rfu)

First pass success rate

(175Rfu)

Off-scale alleles Failures

Partial Profile >1

No Profile

<1 <3 >3

Blood Internal Site 27 1 0 59/60 (98%) 58/60 (97%) 0 0

Blood Site #1 28 0 0 60/60 (100%) 59/60 (98%) 10 18

Buccal Internal Site 28 3 0 57/60 (95%) 56/60 (93%) 3 4

Buccal Site #2 28 3 1 55/59 (93%) 49/59 (83%) 5 4

Buccal Site #3 28 0 1 55/56 (98%) 55/56 (98%) 1 3

Blood and buccal samples on treated paper


Single-Source Protocol – Performance Study

Profiles show good intra-color balance in all dye-channels Data from three external and two internal test sites; Profiles with partial profiles or off-scale alleles were excluded from the analysis

Blood and buccal samples on treated paper

BuccalFTABloodFTA

1.0

0.8

0.6

0.4

0.2

0.0

ICB

STS-Test Site Data: Direct Amplification Samples

Blood Samples

Buccal Samples


On a scale of 1 (highly unlikely) to 5 (highly likely), how likely would your lab utilize amplifying Y-

STRs in direct mode?

1.  Highly Unlikely 2.  Somewhat Unlikely 3.  Uncertain/ Not Applicable 4.  Somewhat Likely 5.  Highly Likely


Close Relative Discrimination with the Yfiler® Plus Kit

Preliminary Investigations


Discrimination between Close Relatives Using Y-STRs

§  Current Y-STR sets are not suited for those situations in which discrimination between close relatives is required

§  Recent studies identified new Y-STRs with higher mutation rate that may help solve this problem −  Rapidly Mutating (RM) Y-STRs

§  Yfiler® Plus kit includes 7 RM Y-STRs in addition to (relatively) slowly mutating Y-STRs


Discrimination between Father-Son Pairs based on Mutations

Yfiler® PPY23 Yfiler® Plus In-House (N=53

Father-Son Pairs) 4 (7.5%) 5 (9.4%) 10 (18.9%)

Budowle Lab (N=95) 6 (6.3%) NA 9 (9.4%)*

*1 Father-Son Pair had 2 mutation events

YFiler® (N=386) PPY23 (N=386) Yfiler Plus-DYS460* (N=331)

27 (7.0%) 44 (11.4%) 58 (>15%)^

NIST Mutation rates in >330 Meioses

* NIST does not have data for the DYS460 marker. ^The Yfiler® Plus RM-STR markers were analyzed in only 331 father-son pairs.


Summary

§  Multiplex core of Yfiler® Plus kit formed by the existing markers in the current Yfiler® Kits to maximize compatibility with existing databases

§  New markers chosen based on gene diversity, mutation rate and copy number −  10 new markers included to maximise haplotype resolution −  7 rapidly mutating markers included to provide potential for discrimination of male

relatives

§  Validation results show: −  Higher number of alleles recovered at low DNA input amounts −  Improved recovery of alleles and intracolor balance in male/female mixtures

containing high female background −  Improved recovery of alleles in inhibited samples −  High first-pass success rate with direct amplification protocol with single-source

samples

§  Preliminary data shows higher capability to discriminate between close relatives with the Yfiler® Plus kit as compared to the Yfiler® kit


Getting ready for the Yfiler® Plus Kit


Be Part of the Yfiler® Plus Kit Project

Become a Test Site

•  Support our rigorous development process •  Be the first to evaluate the Yfiler® Plus kit with your challenging samples

Help Us Grow the Population Databases

•  Help other forensic laboratories by sharing your population data

Join the Y-STR Community

•  Be part of the Y-STR discussions with colleagues around the world along with the Life Technologies team


Expansion of Y-STR Haplotype Databases

§  Increase statistical significance by −  Expanding the population groups represented −  Increasing the number of individuals in each population group

Size Haplotype Database

Probative value of Y-STR

Evidence


Yfiler® Plus & Y-STR Haplotype Databases

§  Partnering with forensic laboratories globally to type a comprehensive set of samples with the Yfiler® Plus kit −  Initial Goal: 1,500-2,000 haplotypes available prior to launching

the kit to the broad forensic community

§  Working closely with main reference databases (Y-HRD

& US Y-STR Database) to update databases (both infrastructure and content)

§  The Yfiler® Haplotype Database will continue to exist but

will not be updated with Yfiler® Plus data


Y-STR Community & Discussion Forum §  Web based Y-STR resource

hub and information center developed to: −  Provide the most updated

information in the area of forensic Y-STR analysis

−  Facilitate communication between Life Technologies and its customers

−  Help forensic scientists around the world collaborate and stay in touch

www.lifetechnologies.com/ycommunity

development of a next generation y-str multiplex for forensic applications

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