# Presentation: 333209

## International Society of Forensic Genetics
August 30, 2001
John Butler 	      Rich Schoske 	   Pete Vallone

- August 30, 2001
- John Butler 	      Rich Schoske 	   Pete Vallone

- Highly Multiplexed Assays for Measuring Polymorphisms on the Y-Chromosome

## We need:

**There is a growing interest in the Y-chromosome to aid forensic and paternity testing...**
- (>50 presentations here at ISFG on Y markers)

- http://www.ncbi.nlm.nih.gov/genome/guide/

- Human Genome

**1     2     3    4     5     6     7     8     9    10   11   12**

**  ****13   14   15  16   17   18   19   20   21   22   X     Y**

_**We need**_**:**
**Population data**
**Multiplex assays**
**Standards**

## http://www.ystr.org/europe/

**European Y-STR Haplotype Reference Database** Created by Sascha Willuweit and Lutz RoewerInstitute of Legal Medicine, Humboldt-Universität Berlin, Germanyin cooperation with Michael Krawczak (Cardiff), Manfred Kayser (Leipzig) and Peter de Knijff (Leiden)

- This database has been accessed **14809** times since 01/01/2000. Last haplotype entry **3/26/2001**
- Current state of the database:		**45** European population samples		**5529** minimal haplotypes, **2196** of these are extended haplotypes

- n = 850 mt-DNA D-Loop sequences (data kindly provided by the Institute of Legal Medicine Magdeburg, Germany)n = 2196 extended European haplotypes logged in the database

## Standard Way to Type “Extended Haplotype”

- GDB primers
- 2-3 multiplexes for minimal haplotype
- DYS385 run separately sometimes
- YCAII run separately to obtain extended haplotype
- Different PCR conditions
- Primers often require titrations (10 fold difference in amounts) in order to establish reasonable balance between Y STR PCR products

**Published primers were not designed to work together (originally used in single-plex PCRs)**

## Commonly Used Y STR Multiplex Assay

- DYS19

- 389II

- 389I

- 390

- _Primer_   _Amounts__	__Dye_
- Y19	0.4 M	TET
- Y389	0.12 M	TET
- Y390	0.12 M	FAM
- Y393	0.2 M	HEX

*Gusmão et al. 1999            **(Forensic Sci Int, vol. 106, pp. 163-172)*

**5-plex**

- 393

**Higher level multiplexes are needed to improve the power of discrimination for Y chromosome DNA tests**

**Inefficient use of space across size range and dye color**

## Assay Development Goals

- Single tube amplification of all loci in minimal (9-loci) or extended (11-loci) European Y STR haplotypes
- Incorporation of additional polymorphic markers recently published
- No female amplification products (avoid X chromosome homology)
- Spacing between loci in same color to allow additional undiscovered alleles to be accommodated
- Similar concentration of primers to produce balanced amplification products
- Sensitivity to <100 pg male DNA with 28 cycle PCR

*Discussion with M. Prinz December 2000*

## Development Strategy for Y STR Multiplexes

- Careful definition of allele ranges
- _Literature searches_ of over 200 papers to locate all known alleles
- _Evaluation of diverse population samples_ to search for rare alleles (M. Hammer cell lines)
- Permits markers to be packed together more closely in a single dye color
- Avoiding polymorphic nucleotides in primer binding sites
- _Alignment of multiple GenBank entries_
- Permits primers to be designed with less risk of null alleles
- Incorporation of newly developed NIST multiplex assay design and testing tools

## Tools for Multiplexing

- Primer design software
- Visual Basic programs to check potential primer dimer formation
- Quality control testing of primers
- Butler *et al.* (2001) *Forensic Sci. Int.* 119: 87-96
- Rapid multiplex testing
- Butler *et al.* (2001) *Fresenius J. Anal. Chem.* 369: 200-205

## YCC Panel Sample Typed with Reliagene Y STR Kit

## NIST Y STR 10-plex

- A7.1

- H4

- DYS391

- DYS438

- DYS437

- DYS392

- DYS435

- DYS439

- DYS19

- DYS436

**NIST Y STR 10-plex**

*Primers were redesigned for most of these loci in order to keep the PCR products under 200 bp so that degraded DNA could be more successfully typed.*

*Designed by Christian Ruitberg*

- Presented at Promega meeting October 2000

## Slide 11

## NIST Y-STR 20plex

**426**

**385II**

**385I**

**393**

**YCAII**

**390**

**392**

**A7.1**

**447**

**391**

**  ****19**

**437**

**389I**

**389II**

** ****438**

**448**

**H4**

**388**

**439**

- GS500 LIZ internal sizing standard

**NIST Y-STR 20plex**
*Developed by Rich Schoske and John Butler*

**Incorporates all loci in the European Y STR “extended haplotype” plus a number of other useful markers**

## Y STR 20plex utilizing 5 dye chemistry

***Y STR 20plex utilizing 5 dye chemistry***

- Genotyper result

## Schematic of Loci in NIST Y STR 20-plex

**438**

**391**

**389I**

**437**

**19**

**389II**

**6FAM (blue)**

**393**

**390**

**385 I/II**

**426**

**YCAII**

**VIC (green)**

**A7.1**

**H4**

**439**

**392**

**388**

**NED (yellow)**

**447**

**448**

**PET (red)**

*Details will be presented at the Promega meeting in October 2001*

- Utilizes 5-dyes

**Same dyes as new ABI Identifiler****TM**** kit**

**Schematic of Loci in NIST Y STR 20-plex**
***Designed by Richard Schoske***

***Loci outlined in red have new primers***

## Schematic of Loci in NIST Y STR 20-plex

**391**

**389I**

**437**

**19**

**389II**

**393**

**390**

**385 I/II**

**A7.1**

**H4**

**439**

**392**

- 91-119

- 133-169

- 184-196

- 214-250

- 257-289

- 108-136

- 191-227

- 240-304

- 99-119

- 129-143

- 204-224

- 242-272

**7**

**16**

**25**

**33.2**

**7**

**16**

**18**

**27**

**7**

**23**

**9**

**16**

**7**

**14**

**14**

**17**

**9**

**14**

**A**

**E**

**6**

**16**

**7**

**12**

**438**

- 299-334

**6**

**13**

**388**

**11**

**17**

- 153-171

**YCAII**

- 145-165

** ****17**

**27**

**426**

-       97

**A**

**?**

**447  **

**448**

- 206-241

- 273-309

**22**

**17**

**23**

**29**

**VIC**

**PET**

**NED**

**6FAM**

**Schematic of Loci in NIST Y STR 20-plex**
***Designed by Richard Schoske***

## Advantages of Y STR 20plex

- Single amplification of “extended haplotype”
- European loci: 19, 385 I/II, 389I, 389II, 390, 391, 392, 393, YCAII a/b
- Additional loci: 426, A7.1, H4, 388, 437, 438, 439, 447, 448
- Sensitive to <500 pg with 28 cycle PCR
- Male-specific with >100X female DNA
- 10 loci with amplicons less than 200 bp in size to aid results with degraded DNA

## Mass spec primers designed for DYS391

**Mass spec primers designed for DYS391**

- Allele with 9 TCTA repeats = 99 bp

## DYS391 Primer Improvements

**Y**

**X**

- *

- *

- *

- *

- *

- *

- *

- Significant homology exists between X and Y
- We have designed primers to anneal to regions that only appear on the Y chromosome (target X deletion regions)
- Primers also work well to produce small PCR products that can be readily analyzed by mass spec

## Power of Discrimination

- _Loci Tested_

- DYS385 (1)
- 19, 389I/II, 390, 393 (5)
- Reliagene Y-Plex 6 kit (6)
- Minimal haplotype (9)
- Extended haplotype (11)
- NIST Y STR 20plex

- _Unique Haplotypes_	_Discrimination*_

- 18
- 62
- 79
- 83
- 87
- 89

*Tested against 97 male DNA samples including YCC panel*

- *results based on different combination from subsets from 20plex testing results...
**http://dna-view.com/haplofreq.htm** (Charles Brenner; chance of obtaining a non-unique haplotype)

- ~1:56
- ~1:210
- ~1:470
- ~1:600
- ~1:900
**~1:1100**

## Equal amount of Female DNA     (no amplification)

- Equal amount of **Female DNA**     (no amplification)

**Male DNA**

**Result obtained with ABI 3100 capillary array instrument**

- Red peaks are LTI 50-500 ROX internal size marker

**Male-Specific Amplification with Y STR Megaplex (16plex with 4 dye chemistry)**

## 7680 data points in 24 hours using new Y STR 20plex

**High-throughput Y STR Typing on the ABI 3100 (16-capillary array)**

**7680 data points in 24 hours using new Y STR 20plex**

- Capability to double current size of European Y STR database in less than one week with one instrument

## 28 cycle PCR with sensitivity of <0.5 ng

**Sensitivity Study with new NIST Y STR 16-plex**

## Plans with our Y STR Megaplex

- Currently being tested in 2 other labs
**M. Prinz** -- forensic samples with 4 dye 16plex
**M. Hammer** -- population studies with 5 dye 20plex
- Concordance studies with YCC sample panel
- Allele sequencing...developmental validation
- Presentation at ISFG and Promega meeting
- Publications with complete details
- Commercializing into kit form?

## Haplogroups produced by Y SNP genotyping

- Underhill *et al.* (2000) *Nature Genetics* 26: 358-361

## MS Data from Y SNP Marker M96

**5154 Da**

**5154 Da**

**5467 Da**

**5427 Da**

- Depurination of primer

- Sample A

- Sample B

**MALDI-TOF MS data can be collected in 5-10 seconds**

- Vallone and Butler, Analysis of SNPs by MS, *Encyclopedia of Mass Spectrometry, in press*

## Mass Spec Y SNP Multiplex Assay

- (M9, M45, M89, and M96)

**P**

**Ex**

**Ex**

**Ex**

**Ex**

**P**

**P**

**P**

## Luminex Bead Y SNP Multiplex Assay

- CM55 Test Sample (H1)

- (9 Y SNPs plus amelogenin)

*Result from Invitrogen beta-test kit*

## Male-Specific Multiplex PCR at 17 Y SNP Loci

- 17 plex PCR

**Female sample (K562)**

**Male sample (AM209)**

- 20 bp ladder

- 200 bp

- 300 bp

- 400 bp

- CE Separation Time (min)

**Remaining primers**

_**Y SNPs Amplified**_
- M2 A/G	 	M60 +T
- M3 C/T		M67 A/T
- M9 C/G		M69 T/C
- M13 G/C	M89 C/T
- M26 G/A	M96 G/C
- M35 G/C	M119 A/C
- M42 A/T	M122 T/C
- M45 G/A	M145 G/A
- M55 T/C

## 5 male samples selected to represent diverse combination of alleles

- SRM 2395 Candidates (Y STR 20plex Data)

*Female test sample yielded no amplification*

## Genotyper Results with Reliagene Y Plex 6 Kit on 5 male candidate samples for SRM 2395

- Genotyper Results with Reliagene Y Plex 6 Kit on 5 male candidate samples for SRM 2395

## STRBase

**Short Tandem Repeat DNA Internet Database**

- http://www.cstl.nist.gov/biotech/strbase

- _Y-Chromosome STR Information Available_
- Over 200 publications on Y STRs & SNPs cataloged
- Allele information on 16 Y STR loci
- Downloadable PowerPoint on Y STRs and Y SNPs
- Links to other Y-chromosome sites
- Information on new Y STR multiplexes developed at NIST

## NIST Work with Y Markers

- Y STR Multiplex Assays (Y STR 20plex)
- Y SNP Multiplexes and Marker Evaluation with MALDI-TOF MS and other technologies
- Development of Y Chromosome Standard Reference Material (SRM 2395)
- Information on Y chromosome markers is being made available through STRBase

## Acknowledgments

- _NIST Personnel_:
- John Butler (Project Leader)
**Pete Vallone**
**Margaret Kline**
- Jan Redman
**Rich Schoske** (AU)
- Gordon Spangler (AU)
- Christian Ruitberg (RPI)
- Dave Duewer (Anal. Chem.)

_**Funding**_**:  **
**National Institute of Justice Grant #97-LB-VX-0003**
**Interagency Agreement between NIJ and NIST Office of Law Enforcement Standards**

- _Collaborators_:
- Mike Hammer and Alan Redd (U. Arizona) for Y STR sequences and samples
- Mecki Prinz (NYC OME)
- Dave Carlson (Invitrogen) on Y SNP work with Luminex beads

- Lisa Forman (NIJ) for support and encouragement