Jack Ballantyne, Erin HansonNational Center for Forensic Science, UCF

In the US, an estimated 19.3% of women and 1.7% of men have been raped during their lifetimes

-CDC Sept 2014

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• No suspect identified• Analysis not requested by prosecutor• Lab not accepting new evidence due to backlog• Officer uncertain of usefulness• Lab was unable to produce timely results• Incident took place > 72 hours ago

Worse than reported: excuses why some

rape evidence is not submitted?

� some rape victims report the incident > 3 days after the incidente.g. incapacitation by drink or drugs/ acquaintance rape

� in many jurisdictions, no intimate samples collected from rape victims if reported > 3 days

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• myth or fact? sperm normally only persist in observable numbers in the post-coital vagina up to 3 days after intercourse

• standard DNA-STR profiling is not possible in late reported intimate post coital samples

• male DNA profile recovery success diminishes rapidly as the post-coital interval is extended: > 24-48h difficult >72 h not normally possible• BUT from the literature sperm (albeit few in number) may be

detectable in the vaginal canal 7-10 days after intercourse

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1998: maybe Y chromosome analysis can help resolve rape

cases that standard DNA analysis can’t?

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• Only found in males• Can ‘ignore’ any female

DNA from victim• In mixed M/F samples

sometimes standard DNA analysis cannot detect the M component• Although discrimination not

as good as autosomal DNA-STR testing

• Use Y!!!

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• Prescribes the use of Y-STRs in certain case circumstances:

“Y-STR typing is also used in lieu of autosomal typing for the

detection of male DNA in mixtures that contain an overabundance

of female DNA. Given that under certain conditions a male minor

contributor in a mixture of female:male DNA may only be

detectable by Y-STR typing, laboratories should pursue Y-STR

analysis as the most appropriate means of detecting a male

contributor(s) in some forensic samples”.

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“Quantitative PCR that enables the estimation of the

quantities of both male and female DNA is an important

tool for the determination of an amplification strategy

that maximizes the potential for determining the male

component(s) of a mixture”.

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T. Large AutosomalT. Small Autosomal

Total Human QuantAlso

Quality (Degradation) Index

T. Y

Male DNA Quant

F/M Ratio

Autosomal STR analysis(low ratio?)

orY-STR analysis

(high ratio?)

Yfiler™ Plus kit

Improved sensitivity?More robust?Better discrimination

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Pater Current Controlled Trials in Cardiovascular Medicine 2005 6:5 doi:10.1186/1468-6708-6-5

Quant threshold?

+ profilesno profiles

Labs can determine their own LOQ if desired

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Yfiler™ Plus kit

� All 17 Yfiler™ loci plus 10 new loci (7 RM loci)� Improved results with challenging samples

� Direct amp protocol for single source samples� Shorter amp times

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New Y-STR Kits-more loci-more discrimination-better chemistry-RM loci: higher probability of discriminating patrilineal relatives

• SensitivitySensitivitySensitivitySensitivity

• Discriminating profiles Discriminating profiles Discriminating profiles Discriminating profiles with as little as 31.25 with as little as 31.25 with as little as 31.25 with as little as 31.25 –––– 62.5pg male DNA62.5pg male DNA62.5pg male DNA62.5pg male DNA

• AvgAvgAvgAvg No. of Alleles = 17 and 25 alleles, respectivelyNo. of Alleles = 17 and 25 alleles, respectivelyNo. of Alleles = 17 and 25 alleles, respectivelyNo. of Alleles = 17 and 25 alleles, respectively

• InhibitorsInhibitorsInhibitorsInhibitors

• Full profiles in presence of high concentrations of Full profiles in presence of high concentrations of Full profiles in presence of high concentrations of Full profiles in presence of high concentrations of hematinhematinhematinhematin(20,200(20,200(20,200(20,200µµµµM) and M) and M) and M) and humichumichumichumic acid (10,100 acid (10,100 acid (10,100 acid (10,100 ngngngng////µµµµl) l) l) l)

• cccc.f. .f. .f. .f. YfilerYfilerYfilerYfiler inhibited (no male profiles) at higher inhibited (no male profiles) at higher inhibited (no male profiles) at higher inhibited (no male profiles) at higher concconcconcconc

• MixturesMixturesMixturesMixtures

• Full male profiles in 1:1000 and 1:4000 male/female mixturesFull male profiles in 1:1000 and 1:4000 male/female mixturesFull male profiles in 1:1000 and 1:4000 male/female mixturesFull male profiles in 1:1000 and 1:4000 male/female mixtures

• Minor male profile detected in 1:8 Minor male profile detected in 1:8 Minor male profile detected in 1:8 Minor male profile detected in 1:8 male:malemale:malemale:malemale:male mixturesmixturesmixturesmixtures

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• Cervicovaginal swabs collected from a donor couple• 1, 2 and 3 days after intercourse

• Separate acts

• Pre-coital swabs collected for each interval to ensure no prior male DNA present

• Donors abstained for at least 7 days between collections

• Differential (organic) extraction performed

• 3 washes of sperm pellet after removal of non-sperm fraction

• Sample elution volume = 50 µµµµl

• Quantifiler® Trio quantitation (2µµµµl, 4% of extract)

• Amplifications

• Autosomal STRs – GlobalFiler® (29 cycles)

• 1ng when possible; up to 10µµµµl for samples with low or undet quant

• Y-STRs – Yfiler™ Plus kit (30 cycles)

• 1ng when possible; up to 10µµµµl for samples with low or undet quant

• All fractions amplified with both kits for this study regardless of quant value

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Observed typing results are consistent with expecte d results based on Quantifiler® Trio Results

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GlobalFiler ®

1 day

NSP – female only

SP – major male (minor female)

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Yfiler™ Plus1 day

NSP (27/27) SP (27/27)

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GlobalFiler ®

2 dayNSP – female only

SP – mixed (~1:1)

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Yfiler™ Plus2 day

NSP (0/27) SP (27/27)

• 28 28 28 28 of 69 UTHSC kits of 69 UTHSC kits of 69 UTHSC kits of 69 UTHSC kits (~40%)(~40%)(~40%)(~40%)

• Collaborative study on extended interval samplesCollaborative study on extended interval samplesCollaborative study on extended interval samplesCollaborative study on extended interval samples

• 4 4 4 4 day, 7 day and 9 day samplesday, 7 day and 9 day samplesday, 7 day and 9 day samplesday, 7 day and 9 day samples

• NonNonNonNon----differential extracts onlydifferential extracts onlydifferential extracts onlydifferential extracts only

• Combined all fractions for each sampleCombined all fractions for each sampleCombined all fractions for each sampleCombined all fractions for each sample

• Cervix/fornix and standard/concentrated fractions Cervix/fornix and standard/concentrated fractions Cervix/fornix and standard/concentrated fractions Cervix/fornix and standard/concentrated fractions

• To include ‘all available’ DNA To include ‘all available’ DNA To include ‘all available’ DNA To include ‘all available’ DNA

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• Can we improve our analysis of extended interval post coital samples using the integrated/modular Quantifiler® Trio/Yfiler™ Plus system?

Not a typical validation study!

Very challenging (and unusual) sample set!

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• Our data clearly demonstrates improved profile recovery using Y-specific nested PCR pre-amplification

• Cannot be readily be performed at this time due to proprietary primer mix

• Next generation Y-STR kits?

• Eliminate need for pre-amplification?

• improved sensitivity and robustness

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333:1 to 4,685,117:1AutoSTR not possible!

N= 84 (28 x 3)

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N =84

29Sample counted if any alleles recovered >1 allele

N=28 x3

27/27

2719ng total in amp; M/F 1: 5,056

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1730ng total in amp; M/F 1:1,785,058

0/27

• Quantitation System

• Excellent correlation between male quantitation and profile recovery

• Negative quant (threshold based) means negative/unusable Yfiler™ Plus kit

• Observed autosomal/Y results consistent with expected based upon quant and

F/M ratio

• Yfiler™ Plus kit

• Sensitive chemistry

• If >100 pg male, most full profiles

• Results obtained with high quantities of background female DNA (up to µµµµg)

• Many ‘Usable profiles’ F/M ranging from 333:1-100,000:1

• Y-STR profiles using YFP can be obtained at least up to 9 days after intercourse

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• Laboratories should provide or arrange to provide Y -STR typing services in addition to standard DNA analysi s

• Jurisdictions should collect samples from samples a t least up to 10 days after intercourse

• What is the limit of recovery of DNA profiles from the semen donor (surely > 9 days)?

• Recovery of standard autosomal DNA profile > 4 days ?• Other biological and socio-economic factors effecti ng

recovery of male DNA from the cervico-vaginal tract• More loci-NGS- complete male differentiation

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The end crowns all, and that old common arbitrator, Time, will one day end it.

Shakespeare

Thank you for your attention!

Acknowledgements: Robert Green, Allison Holt, Christina Bormann Chung, Julio Molero, Melissa Kotkin (a.k.a Missy)Tamyra Moretti (FBI)

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