Lutz Roewer, Dept. Forensic Genetics The golden age of forensic Y-STR typing – new markers, new databases, new technologies Lutz Roewer, Dept. Forensic Genetics Institute of Legal Medicine and Forensic Sciences, Charité – Universitätsmedizin Berlin ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

PowerPlex Y (12 Loci) Promega Y-Filer (17 Loci) Applied Biosystems Minimal Haplotype PowerPlex Y (12 Loci) Promega Y-Filer (17 Loci) Applied Biosystems PPY23 (23 Loci) Promega YFiler plus beta (27 Loci) Applied Biosystems Commercial + add. Y-STRs (45 loci) DYS19 x DYS389I DYS389II DYS390 DYS391 DYS392 DYS393 DYS385ab (2)   DYS437 DYS438 DYS439 DYS448 DYS456 DYS458 DYS635 YGATAH4 DYS570 DYS576 DYS533 DYS481 DYS549 x  DYS643  x DYS460 DYS627 DYS518 DYS449 DYF387S1ab (2) DYF399S1 (3) DYF403S1 (3) DYF404S1 (2) DYS526 DYS547 DYS612 DYS626 DYS464 (4) ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Really fast mutating Y-STR markers are very rare Ballantyne KN, Goedbloed M, Fang R, Schaap O, Lao O, Wollstein A, Choi Y, van Duijn K, Vermeulen M, Brauer S, Decorte R, Poetsch M, von Wurmb-Schwark N, de Knijff P, Labuda D, Vézina H, Knoblauch H, Lessig R, Roewer L, Ploski R, Dobosz T, Henke L, Henke J, Furtado MR, Kayser M (2010) Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications. Am J Hum Genet 87(3):341-53. ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Family analysed with 45 markers – patrilineage resolved with 2 mutations 576 3 8 9 I 4 II 1 481 549 533 438 437 570 635 390 439 392 643 393 458 5 ab 6 G A T H 2 7 460 518 449 S 526a 526b 626 S1 612 547 F DYS391 µ=2.54 x 10-3 DYS626 µ=1.22 x 10-2 ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Yfiler® Plus 25 marker, 2 bilocal 6 dye matrix ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Ranking of mutation rates (Yfiler® Plus) Loci Mutation Rate [95% CI] Meioses Position[MutRate] Group[MutRate] dys438 2,96E-04 10122 1 slow dys392 4,04E-04 14867 2 dys393 1,09E-03 13713 3 dys437 1,19E-03 10101 4 dys448 1,65E-03 6678 5 dys390 2,06E-03 15061 6 medium dys385 2,30E-03 25620 7 dys19 2,32E-03 15539 8 ygatah4 2,47E-03 7709 9 dys391 2,54E-03 14935 10 dys389i 2,68E-03 13788 11 dys635 3,72E-03 7525 12 dys389ii 3,78E-03 13759 13 dys456 4,19E-03 14 dys481 4,97E-03 1744 15 dys533 5.01E-03 1730 16 dys439 5,35E-03 10096 17 dys460 6,22E-03 1717 18 dys458 6,74E-03 6677 19 dys518 1,84E-02 1556 20 fast dyf387S1ab 1,59E-02 1804 21 dys576 1,43E-02 1727 22 dys570 1,24E-02 1426 23 dys627 1,23E-02 1766 24 dys449 1,22E-02 1617 25 ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Continental proportions of distinct haplotypes determined with slow, middle and fast mutating marker sets ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Increased discrimination ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Local Y-STR diversity differs between continents ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Growth of the YHRD (2000-2014) All haplotypes Haplotypes 17 loci Haplotypes with Y-SNPs Haplotypes 23 loci ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Count Estimates Augmented Choose the most informative haplotype format 17 loci 0/19,593 0/71,246 1.4 x 10-5 5.1 x 10-5 260/125,700 2.1 x 10-3 23 loci 9 loci Count frequency Choose the most informative haplotype format in the largest available database! ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Frequency estimation methods Counting methods Methods taking evolutionary distance between haplotypes into account Augmented counting (1/n+1) Counting with database inflation (Brenners κ) Surveying method (Krawczak) Coalescence based estimation (Caliebe) Discrete Laplace method (Andersen) ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Case-sensitive workflow (touch DNA, mixture expected) Autosomal analysis Mixed female/male profile Autosomal male profile clearly identifiable Report National Police Database Y chromosomal analysis Male profile Direct matching possible? Database (YHRD) frequency ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

National Police Database Charité Pilot study to evaluate the workflow: 40 sexual crimes with only „touch“ DNA (2011) 40 cases 8 cases Individual male autosomale profile 10 cases Informative YSTR Profile 22 cases inconclusive National Police Database Report with YHRD match statistics Number of informative profiles doubled ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2013

Casework statistics since implementation of the PPY23 kit (08/2012 bis 02/2014) 160 cases (1038 contact stains) autosomal No male admixture observed in the Amelogenin system 30% Additional Y peak in Amelogenin 70% 37.5% informative for the male component Y-chromosomal No Y profile (11%) Y profile observed (89%) informative Y profile 58% 2012 – 21 Vergleichsproben 2013 – 31 Vergleichsproben 29% (=14 delicts) with Y-Profil Single source Y profile (>20 Y-STRs) 39% ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Case example 1: touch stain on fabric in a sexual assault case Autosomale Analyse Y-chromosomale Analyse Kontaktspur, Sexualdelikt Nur ♀-Komponente, ohne Hinweis auf ♂-Beimischung im Amelogenin Vollständiges Y-chromosomales Einzelprofil in 23 loci ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Case example 2: touch stain on fabric in a child abuse case Yfiler + SNaPshot 9/16 Y-STRs detectable Haplogroup I (M170) ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Case example 2 (continuing): Prediction of ancestry Haplogroup I (I2a) can also be reliably predicted from YHRD data ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Case example 3: Serial rape - linking 3 crime scenes DYS 389I 448 389II 19 391 438 437 635 390 439 392 393 458 385ab 456 GATA H4 Case1 13 29 14 10 12 15 24 22 17 11,14 16 Case2 nd Case 3 13+ 19+ 29+ 14+ 10+ 12+ 15+ 22+ 11,14+ 16+ Consensus profile DYS 389I 448 389II 391 437 393 458 385ab 456 13 19 29 10 15 17 11,14 16 Suspect 12 27 11 14 Exclusion of the suspect ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Is NGS the future for complete resolution of male relatives ? ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Next generation sequencing 10 Mb (of 24 MB NRY) sequenced 67 STRs identical 4 SNPs different Potentially long sequence reads more variable than STRs Xue Y, Tyler-Smith C 2009 ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Summary Wider acceptance of forensic Y chromosome analysis due to a new generation of diagnostic kits with high discriminative power and much improved performance Complete resolution of distant relatives in most populations a proportion of close relatives can be differentiated Adaptation of the workflow, otherwise you‘re losing evidence! Necessity to employ statistics (LR) to evaluate the match probability Haplotype databases accomodate the profiles of the new generation kits (www.yhrd.org) Y-STRs are included in different National DNA Databases (USA, Netherlands, Austria) For Forensic or Paternity Use Only. Yfiler is a trademark of Thermo Fisher Scientific and its subsidiaries. ©Charité – Universitätsmedizin Berlin, Dept. Forensic Genetics 2014

Disclaimers For Forensic or Paternity Use Only. Yfiler is a trademark of Thermo Fisher Scientific and its subsidiaries.