Developing multiplex PCR assays for human identity testing - is there overlap with pathogen screening?

1. 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

2. 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

3. 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

4. qPCR Blood Stain Buccal swab DNA Quantitation DNA Extraction Sample Collection & Storage Multiplex PCR Amplification DNA separation and sizing DNA Database Search STR Typing Interpretation of Results Steps in DNA Analysis Steps Involved Usually 1-2 day process (a minimum of ~5 hours) Collection Specimen Storage Extraction Quantitation Biology Multiplex PCR 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 Genetics Interpretation of Results Usage: Human ID Paternity Test Missing Person Mass Disaster Technology

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

6. D21S11 D8S1179 D7S820 CSF1PO D13S317 D3S1358 TH01 D2S1338 D16S539 TPOX VWA D19S433 D18S51 AMEL D5S818 FGA Example Profile of a Short Tandem Repeat Assay Information is tied together with multiplex PCR and data analysis 16 STR loci amplified in a single reaction 0.5 ng of human genomic DNA Identifiler STR Kit from Applied Biosystems

7. ABI PRISM® SNaPshot™ Multiplex System Fluorescently labeled ddNTPs + polymerase SNP Primer is extended by one base unit “tail” used to vary electrophoretic mobility G T Oligonucleotide primer 18-28 bases A 3’ 5’ C G PCR Amplified DNA Template SNP Genotyping Allele-Specific Primer Extension 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

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

9. 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

10. In House Assay Design

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

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

13. 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

14. 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

15. 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

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

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

18. 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

19. 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)

20. 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

21. 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

22. 2 1 4 3 5 6 7 8 10 11 12 9 13 14 15 16 18 17 19 Autosomal 26-plexUnder Development (19plex so far) 6FAM VIC NED PET 1 ng DNA; 30 cycles

23. 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

24. 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

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

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

27. Becky Hill Amy Decker John Butler Jan Redman Dave Duewer Margaret Kline Acknowledgments 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… petev@nist.gov