Y-Screening and Direct Amplification of Sexual Assault Evidence Kit Samples. October 31, 2016 Presented by:

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1 Y-Sreening and Diret Amplifiation of Sexual Assault Evidene Kit Samples Otober 31, 2016 Presented by: Anupama Gopalakrishnan, PhD Promega Corporation Amy MGukian, MSFS, F-ABC Palm Beah County Sheriff s Offie 2013 Promega Corporation Promega Corporation.

2 Y-Sreening and Diret Amplifiation of Sexual Assault Evidene Kit Samples Anupama Gopalakrishnan 2013 Promega Corporation.

3 Casework Diret Kit, Custom The Casework Diret Kit is used for rapid proessing of swabs from asework samples or uttings of sexual assault swabs and stained lothing prior to quantifiation of human DNA using the PowerQuant or Plexor HY System and amplifiation of normalized template using PowerPlex Systems for human STR genotyping. This kit allows low template samples to be rapidly proessed, quantified, and amplified with minimal loss of DNA. For touh DNA or low template asework samples For Y-sreening appliations 2013 Promega Corporation. 3

4 Produt Configuration Kit Size: 250 reations Materials Supplied in Kit: Component Quantity Casework Diret Reagent, 100ml 1 1-Thioglyerol, 75ml 1 5X AmpSolution Reagent, 500ml 1 Water, Amplifiation Grade, 1,250ml 5 Ethylene-Oxide Treated Materials Available Separately: CW Spin Baskets (50/pk) CW Mirofuge Tubes (50/pk) Available for sale in De Promega Corporation.

5 Simple 2-step protool Casework Diret Solution, µl disard Quantifiation using PowerQuant System or Plexor HY Inubate at 70 C/30 Centrifuge for 5 min 2µl No Wash Steps Minimal potential for DNA loss <15µl STR amplifiation using PowerPlex Systems* 2013 Promega Corporation.

6 Compatibility of Casework Diret Lysate Quantifiation System PowerQuant System Plexor HY System 1X AmpSolution Requirement No Yes STR System PowerPlex Fusion 6C PowerPlex Fusion PowerPlex X AmpSolution Requirement No No No PowerPlex ESX 16/ 17 Fast Yes PowerPlex ESI 16/ 17 Fast Yes PowerPlex Y23* No (15µl max vol) 2013 Promega Corporation.

7 Mok Casework Sample Blood on driveway Swabbed 5 days later Exposed to sun and rain 345pg [Auto]=0.023ng/µl [Auto]/[D]= Promega Corporation Promega Corporation.

8 Deision tree Quantify lysates with PowerQuant System or Plexor HY System No DNA deteted DNA present; high Auto/Y DNA present; high Auto/D DNA present; IPC is flagged Auto STR Y-STR Choose another sample Target higher template Dilute sample Purify sample with DNA IQ hemistry 2013 Promega Corporation.

9 Y-SCREENING AND DIRECT AMPLIFICATION OF SEXUAL ASSAULT SAMPLES A Caseworking Laboratory s Perspetive Amy MGukian, MSFS, F-ABC Tehnial Leader Palm Beah County Sheriff s Offie

10 DISCUSSION POINTS: Why Y-Sreen? Palm Beah County Sheriff s Offie study and onlusions Proposed workflow

11 WHY Y-SCREEN? Conventional sreening is laborious and time onsuming Conventional sreening results give analysts little preditive power as to what the DNA profile may look like An absene of seminal fluid/sperm does not prelude the presene of male DNA (e.g. touh ases) The reent passage of test-all sexual assault kit (SAK) legislation is inreasing demands on forensi biology laboratories Imposing time limits for laboratory analysis Often no additional funding for personnel and other resoures

12 WHY PBSO WAS LOOKING AT Y-SCREENING Senate Bill 636 under onsideration by law makers Signed in to law Marh 23, 2016 Into effet July 1, 2016 Laboratories have 120 days to omplete testing on sexual assaults (not just those with SAKs) Looking at the data this would equate to roughly a 100% inrease in sexual assault ase requests Provided no funding for additional resoures

13 PROTOTYPE TESTING Materials and Methods Prototype Casework Diret Kit Began ollaboration with Promega in February 2016 Studies onduted in May 2016 PBSO Methods QIAGEN EZ1 Advaned XL with DNA Investigator Kit Promega PowerQuant PowerPlex Fusion 5C PowerPlex Y23 AB 3500xl (1.2kV, 24 se. injetion)

14 METHOD Prototype Casework Diret Reagent 1-Thioglyerol Remove spin basket Prototype Casework Diret Reagent and 1-Thioglyerol Centrifuge 5 min. Add sample <1/4 swab Inubate 30min at 80 C Lysate is ready for downstream appliations

15 WORTH NOTING.. Extration method is a whole ell extration DNA reovery is not limited by binding apaity of magneti beads Ability to extrat more male DNA in the presene of high amounts of female DNA Also ability to extrat mirograms of DNA! Diret to DNA approah will limit the amount of sample used as seondary sampling may not be required for additional extrations Consider sample size of uttings Targeted ¼ swab for studies

16 SENSITIVITY STUDY: MALE DNA IN THE PRESENCE OF HIGH FEMALE DNA Vaginal swabs spotted with 10µl donor semen serially diluted. ¼ of a swab was proessed with 400µl Prototype Casework Diret Reagent and laboratory s modified differential extration method. Samples were normalized to 300pg template. Normalization was based on autosomal target DNA onentrations for PowerPlex Fusion and based on Y Target DNA onentrations for PowerPlex Y23.

17 [DNA] (ng/µl) [DNA] (ng/µl) 6 Quantifiation of Semen DNA Based on Y Target neat 1:1 1:2 1:4 1:8 Semen Dilution Prototype Casework Diret Kit PBSO Differential Method :8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 1:4096 1:8192 1:16384 Semen Dilution

18 POWERPLEX Y23-PROTOTYPE CASEWORK DIRECT KIT Undiluted 300pg DNA 1:2 300pg DNA 1:4 300pg DNA 1:2, pg DNA 1:8, pg DNA

19 POWERPLEX FUSION 1:64-PROTOTYPE CASEWORK DIRECT KIT µ

20 POWERPLEX FUSION 1:64

21 STUDY: POST-COITAL TIME INTERVALS Prototype Casework Diret Kit ¼ of a swab was proessed with 400µl Prototype Casework Diret Reagent and the laboratory s differential extration method. Samples were normalized to 300pg template. Normalization was based on autosomal target DNA onentrations for PowerPlex Fusion and based on Y target DNA onentrations for PowerPlex Y23.

22 CORRELATION WITH PRESUMPTIVE SCREENING TESTS Post-oital swabs Drop-in = allele deteted not belonging to expeted profile Drop-out = loss of allele (s) from the expeted profile

23 PowerPlex Fusion System-Prototype Casework Diret Kit 5hr 24hr 48hr 72hr 96hr

24 PowerPlex Y23 System-Prototype Casework Diret Kit 5hr 24hr 48hr 72hr 96hr

25 STUDY CONCLUSIONS Fast Lysates an be prepared in less than one hour inluding sample preparation Sensitive Y23 haplotypes obtained from 1:8192 dilutions with high female bakground Potential to bypass traditional sreening methods Confidene in stopping analysis Robust Potential to amplify lysates diretly for autosomal or Y STRs

26 WORKFLOW PROPOSAL SAKs will be proessed as follows: Presumptive aid phosphatase sreening tests will be performed Samples with positive presumptive test results will be extrated with Casework Diret Kit Deisions based on sample type (e.g. vaginal swabs), and time of assault to SAK olletion will have to be fatored in the deision proess Casework Diret Kit proessing will be onduted during evidene sreening/ preservation, not by the DNA analyst Cuttings from relevant swabs from SAKs will be proessed with the Casework Diret Kit Vaginal and/or related swabs Oral swabs (not oral standards) Retal and/or related swabs Body swabs Negative extrats for male DNA will terminate the need for additional proessing

27 WORKFLOW PROPOSAL Positive samples for male DNA will be assigned to a DNA analyst and either proeed: Diretly to autosomal amplifiation Can utilize probabilisti genotyping tools suh as STRmix to deonvolute mixture (an ondition to intimate sample owner) To re-extration with a differential protool When the presene of female DNA is not high enough to obsure the male profile ompletely, but may ause drop-out Diretly to Y-STR amplifiation When the ratio of female to male DNA would not generate an interpretable autosomal profile from the male omponent

28 Confirming the presene of seminal fluid/sperm will be on a request basis Other Potential to use Casework Diret Kit on other ase types and evidene Multiple stains on an item Touh DNA

29 ADDITIONAL STUDIES Studies to examine different sample sizes Neessary to test multiple swabs from eah area olleted? (i.e. test all six vaginal swabs olleted, both oral swabs, et.) Establish quantifiation ut-offs to determine when a sample should proeed diretly to sample re-extration with differential protool to optimize male profile reovery

30 ACKNOWLEDGEMENTS PBSO Jerome Remm Julie Sikorsky Promega Anupama Gopalakrishnan Jeanne Bourdeau-Heller Bob MLaren Ann MaPhetridge

31 QUESTIONS