Developing multiplex PCR assays for human identity testing - is there overlap with pathogen screening? Dr. Peter M Vallone Human Identification Project U.S. National Institute of Standards and Technology SoGAT XX Warsaw, Poland 12-13 June 2007

Our publications and presentations are made available at: Disclaimers Funding: Interagency Agreement 2003-IJ-R-029 between the National Institute of Justice and NIST Office of Law Enforcement Standards Points of view are those of the authors and do not necessarily represent the official position or policies of the US Department of Justice. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible.  In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose. Our publications and presentations are made available at: http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

Human Identity Project at NIST Genotype samples with commercial assays (nucleic acid based) Produce Standard Reference Materials Training and Interlaboratory Studies Develop novel multiplex assays for genotyping

Goals Interest in further understanding of PCR Learn more about the SoGAT mission Building multiplex PCR assays Upper limits of multiplexing? Robust performance Speed up assay development Can this be of use to your community? Further my knowledge of assay design outside of forensic applications

Applications of Human Identity Testing Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Missing persons investigations Military DNA “dog tag” Convicted felon DNA databases Mass disasters -- putting pieces back together Historical investigations and genetic genealogy Involves generation of DNA profiles usually with the same genetic markers and then MATCHING TO REFERENCE SAMPLE

Steps in DNA Analysis Biology Genetics Technology Steps Involved Usually 1-2 day process (a minimum of ~5 hours) Collection Sample Collection & Storage Buccal swab Blood Stain DNA Extraction DNA Quantitation qPCR Specimen Storage Extraction Quantitation Biology Multiplex PCR Multiplex PCR Amplification STR Typing If a match occurs, comparison of DNA profile to population allele frequencies to generate a case report with probability of a random match to an unrelated individual Interpretation of Results Genetics Work on this one… STR Typing DNA separation and sizing Usage: Human ID Paternity Test Missing Person Mass Disaster DNA Database Search Technology Interpretation of Results

What Type of Genetic Variation? Length Variation short tandem repeats (STRs) Sequence Variation single nucleotide polymorphisms (SNPs) insertions/deletions CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT Tetra nucleotide repeat GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC

Example Profile of a Short Tandem Repeat Assay Information is tied together with multiplex PCR and data analysis D8S1179 D21S11 D7S820 CSF1PO D13S317 D16S539 D2S1338 D18S51 TPOX VWA FGA D5S818 AMEL D19S433 TH01 D3S1358 Total cost per sample = $3.87 (Fall 2002) 16 STR loci amplified in a single reaction 0.5 ng of human genomic DNA Identifiler STR Kit from Applied Biosystems

PCR Amplified DNA Template SNP Genotyping Allele-Specific Primer Extension G C A T Fluorescently labeled ddNTPs + polymerase ABI PRISM® SNaPshot™ Multiplex System SNP Primer is extended by one base unit Oligonucleotide primer 18-28 bases 5’ 3’ “tail” used to vary electrophoretic mobility PCR Amplified DNA Template G Animate this slide!!!! Multiplex PCR followed by a clean up step (Exo-SAP) Multiplex primer extension (with SNaPshot reagent mix) Fragments separated and detected on a gel or capillary electrophoresis platform

Autosomal 12-plex SNP Assay 250 pg 125 pg 63 pg 31 pg 1 2 3 4 5 6 7 8 9 10 11 12

Forensic Assays Limited sample (0.5 – 1 ng of genomic DNA) Multiplex (10 or more loci/amplicons in a single reaction) Robust amplification (results must hold up in court) Issues with degradation, inhibition and efficient sample extraction Standardized testing throughout the forensic community

In House Assay Design

Selection of Loci From Characterize sequence Literature Collaborators Sequencing Characterize sequence Map in software (Lasergene) NCBI accession number ~1000 bp/locus

Selection of Loci High quality sequence information Standardize nomenclature Strand orientation Keep track of primer design

Primer Selection Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) Stand alone version on Mac OSX Visual OMP (www.dnasoftware.com) Standard design parameters (Tm ~60oC) Amplicon length (minimize) Restrict primers to flank target region Take advantage of mis-priming libraries to screen primers

Further Primer Screening AutoDimer software Web based version http://yellow.nist.gov:8444/dnaAnalysis/ Stand alone version http://www.cstl.nist.gov/div831/strbase//AutoDimerHomepage/AutoDimerProgramHomepage.htm BLAST http://www.ncbi.nlm.nih.gov/BLAST/ Avoid significant homology with other chromosomal locations and/or organisms Save search results for further review Confirm primer binding sites

Tools for Primer Selection Screens oligos for primer-dimers interactions Provides Tm, DG, etc (oligo calculator) Design primers for ASPE SNP assays Freely available http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do

Simple Oligo Calculator http://yellow.nist.gov:8444/dnaAnalysis/

Primer-Dimer Screening Open to new ideas and additional functionalities http://yellow.nist.gov:8444/dnaAnalysis

Assay Development UV quantitation of primers (ensure reproducibility) Run PCR in singleplex reactions Samples for assay development Well characterized quality – pristine Contain a sampling of sequence variants Sufficient quantities for testing Well characterized template concentration (qPCR) – define sensitivity limits

Determination of DNA Oligomer Concentrations Expected 100 mM Concentrations were estimated by UV Spec readings @260 using extinction coefficients determined from nearest-neighbor values Lot-to-lot variation of oligo concentration makes it difficult to reproduce a multiplex assay

General PCR Conditions Attempt to keep conditions a constant 1 x PCR buffer 1 Unit of polymerase (TaqGold) 2 mM Mg++ 250 mM dNTPs 0.16 mg/mL BSA 0.2 mM of each PCR primer 0.5 – 1 ng of template DNA (80-200 copies of target)

Assay Development Use singleplex data to evaluate multiplex performance Optimization is empirical Balance PCR primer concentration Replace inefficient primers Identify and replace artifact causing primers Integrate the lessons learned back into the informatics/strategy pipeline

Literature from our Mplex Work Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, 297: 53-66. [preprint] Butler, J.M., David, V.A., O’Brien, S.J., Menotti-Raymond, M. (2002) The MeowPlex: a new DNA test using tetranucleotide STR markers for the domestic cat. Profiles in DNA, Promega Corporation, Volume 5, No. 2, pp. 7–10. http://www.promega.com/profiles/502/ProfilesInDNA_502_07.pdf Butler, J.M., Schoske, R., Vallone, P.M. Highly multiplexed assays for measuring polymorphisms on the Y-chromosome. (2003) Progress in Forensic Genetics 9 (Brinkmann, B. and Carracedo, A., eds.), Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1239, pp. 301-305. Butler, J.M., Shen, Y., McCord, B.R. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci 48(5) 1054-1064. Butler, J.M., Schoske, R., Vallone, P.M., Kline, M.C., Redd, A.J., Hammer, M.F. (2002) A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci. Int. 129: 10-24. Butler, J.M., C.M. Ruitberg, Vallone, P.M. (2001) Capillary electrophoresis as a tool for optimization of multiplex PCR reactions, Fresenius J. Anal. Chem. 369: 200-205. Coble, M.D. and Butler, J.M. (2005) Characterization of new miniSTR loci to aid analysis of degraded DNA. J. Forensic Sci. 50: 43-53. Devaney, J.M. Pettit, E.L., Kaler, S.G., Vallone, P.M., Butler, J.M., Marino, M.A. (2001) Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography. Anal. Chem. 73: 620-624. Just, R.S., Irwin, J.A., O'Callaghan, J.E., Saunier, J.L., Coble, M.D., Vallone, P.M., Butler, J.M., Barritt, S.M., Parsons, T.J. (2004) Toward increased utility of mtDNA in forensic identifications. Forensic Sci. Int. 146S: S147-S149. Menotti-Raymond, M.A., David, V.A., Wachter, L.A., Butler, J.M., O’Brien, S.J. (2005) An STR forensic typing system for genetic individualization of domestic cat (Felis catus) samples. J. Forensic Sci. 50(5): 1061-1070. Schoske, R., Butler, J.M., Vallone, P.M., Kline, M.C., Prinz, M., Redd, A.J., Hammer, M.F. (2001) Development of Y STR megaplex assays. Proceedings of the Twelve International Symposium on Human Identification 2001, Promega Corporation. http://www.promega.com/geneticidproc/ussymp12proc/contents/butler.PDF Schoske, R., Vallone, P.M., Ruitberg, C.M., Butler, J.M. (2003) Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal. Bioanal. Chem., 375: 333-343. Schoske, R. (2003) The design, optimization and testing of Y chromosome short tandem repeat megaplexes. Ph.D. dissertation, American University, 270 pp. Schoske, R., Vallone, P.M., Kline, M.C., Redman, J.W., Butler, J.M. (2004) High-throughput Y‑STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays. Forensic Sci. Int. 139: 107-121. Vallone, P.M., Just, R.S., Coble, M.D., Butler, J.M., Parsons, T.J. (2004) A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int. J. Legal Med., 118: 147-157. Vallone, P.M. and Butler, J.M. (2004) Multiplexed assays for evaluation of Y-SNP markers in U.S. populations. Progress in Forensic Genetics 10, Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1261, 85-87. Vallone, P.M. and Butler, J.M. (2004) Y-SNP typing of U.S. African American and Caucasian samples using allele-specific hybridization and primer extension. J. Forensic Sci. 49(4): 723‑732. Vallone, P.M., Decker, A.E., Butler, J.M. (2005) Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African American, and Hispanic samples. Forensic Sci. Int. 149: 279-286. Vallone, P.M., Decker, A.E., Coble, M.D., Butler, J.M. (2006) Evaluation of an autosomal SNP 12-plex assay. Progress in Forensic Genetics 11, Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1288, 61-63. Vallone, P.M., Fahr, K., Kostrzewa, M. (2005) Genotyping SNPs using a UV photocleavable oligonucleotide in MALDI-TOF MS. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, 297: 169-178. Multiplex Assays Developed at NIST Various Y-SNPs (6-plexes) Y-STR 10-plex & 20-plex Mitochondrial SNP 11-plex Autosomal SNP 12-plexes Coming soon: Autosomal STR 26-plex http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm

Autosomal 26-plex Under Development (19plex so far) 10 11 12 13 14 15 16 17 18 5 4 3 2 7 6 8 19 6FAM VIC NED PET 1 ng DNA; 30 cycles

Rapid PCR with Short Amplicon STRs 1 ng of genomic DNA Initial results amplifying a STR 3-plex in 36 minutes Could show promise for rapid screening

Areas of Concern/Differences Speed ~1 day Extraction 1 hour Quantitation 1 hour PCR ~2.5 hours Capillary/Gel separation 1 hour Faster mutation rate – unique variants (or closely spaced sites) We use qPCR for quantitation not detection, but multiplex assay design strategy may apply

Mitochondrial SNPs Closely spaced polymorphisms Haplotypes will vary from person to person

Standard Reference Materials http://www.cstl.nist.gov/biotech/strbase/srm_tab.htm Traceable standards to ensure accurate measurements in our U.S. crime laboratories SRM 2391b – CODIS STRs SRM 2392-I – mtDNA SRM 2395 – Y-STRs SRM 2372 – DNA quantitation Helps meet DAB Std. 9.5 and ISO 17025 Update materials for new loci Lab 1 Lab 2 Calibration with SRMs enables confidence in comparisons of results between laboratories Standards Reference Material

Summary Evolving strategy for developing multiplex PCR assays Robust assay development assists in interlaboratory testing What aspects can be applied to pathogen screening?

Acknowledgments petev@nist.gov Funding from interagency agreement 2003-IJ-R-029 between the National Institute of Justice and the NIST Office of Law Enforcement Standards NIST Human Identity Project Team – Leading the Way in Forensic DNA… John Butler Margaret Kline Amy Decker Becky Hill Dave Duewer Jan Redman petev@nist.gov

SRM 2372 Human DNA Quant 3 genomic DNA samples highly purified Certified for UV absorbance assume 1 OD = 50 ng/uL of genomic DNA Determine performance (is there bias) Commercial qPCR in house qPCR assays NIST SRM 2372 Human DNA Quantitation Standard Assists with the calibration of commercial and in house standards Monitor lot to lot variation of qPCR standards Candidate 2372 materials were sent out for interlaboratory testing of various qPCR methods in the community before release

Human Identity Testing NIST SRM 2391b PCR-Based DNA Standard Cell Lines and Genomic DNAs Ensures accurate and reproducible testing throughout the human ID community Samples extracted (methods vary) need ~1ng of genomic DNA inhibitors, degradation Multiplex PCR (commercial kits) no in house assays microsatellites 16 loci in a single reaction high degree of reproducibility Protocols for PCR the same across the community Products separated on gel or CE instrumentation Protocols similar across the community Data review Using the same software package Interpretation guidelines

Two Adjacent Mitochondrial SNPs 16223 (C/T) and 16224 (A/G) Relative Intensity m/z 16223 16224 Initial control region 10plex data 16223t primer extension product 16224b primer extension product 297 Da ddA 288 Da ddT

Single PCR product containing 5 polymorphisms Multiple PCR products containing 5 polymorphisms