development of a next generation y-str multiplex for forensic applications
TRANSCRIPT
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
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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
3 Life Technologies™ |
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
4 Life Technologies™ |
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
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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.
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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
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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
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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
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Yfiler® Plus Kit Development 007 Control DNA Profile
1ng of male 007 control DNA amplified for 30 cycles
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Yfiler® Plus Kit Development Direct Amplification of Blood on Treated Paper
1.2 mm FTA® disc amplified for 27 cycles
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Yfiler® Plus Kit Development Direct Amplification of Buccal on Treated Paper
1.2 mm FTA® disc amplified for 28 cycles
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Yfiler® Plus Allelic Ladder
Alleles Virtual Bins Yfiler® Kits 137 72 Yfiler® Plus Kit 345 179
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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
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Yfiler® Plus Kit Development Instrument Compatibility
3500xL 3130xl*
*6-Dye Compatibility with Data Collection Software v4 & 6-Dye module
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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)
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Validation Studies
Summary of Results
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Internal VTS Data
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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.
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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
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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
27 Yfiler PlusPPY23Yfiler
Kit
16.3333
23
27
17
23
27
Mixture Study: Alleles recovered
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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
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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
27 Yfiler PlusPPY23Yfiler
Kit
0
23
27
15.75
23
27
0
23
27
16
23
27
Inhibition Study: Alleles recovered
27
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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
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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
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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
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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
36 Life Technologies™ |
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
37 Life Technologies™ |
Close Relative Discrimination with the Yfiler® Plus Kit
Preliminary Investigations
38 Life Technologies™ |
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
39 Life Technologies™ |
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.
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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
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Getting ready for the Yfiler® Plus Kit
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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
43 Life Technologies™ |
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
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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
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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