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June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addresspqPresentation Outlineheterochromatin State of the Y Chromosome:New Advances and State of the Sciencefor Y Chromosome DNA TestingJohn M. Butler, Ph.D.Canadian Forensic DNA Technology WorkshopToronto, OntarioJune 8, 2005“State of the Y STR Assay” in June 2000From J.M. Butler talk June 1, 2000 at CHI “DNA Forensics” meeting (Springfield, VA) A number of multiplex reactions have beenreported in the literature but Y STR multiplexeshave not reached their potential Very little PCR optimization to-date (most work hasbeen done with the original PCR primer sequences) No commercial Y STR kit exists yet (therefore thesemarkers remain inaccessible to the general forensic DNAcommunity) New Y STR markers are becoming availablewhich will greatly improve the power of discriminationbetween unrelated individuals (e.g., DYS385) and thesewill need to be incorporated into future multiplex setsAdvantages of the Y-ChromosomeCharacteristics of Core Y-STR LociAvailable Y-STR KitsAvailable Y-STR DatabasesY-STR Population StudiesStatistics with Y-STR HaplotypesNew Y-STRsLocus Duplication and DeletionY-SNPsWhat has happened in the past 5 years “Full” Y-chromosome sequence became available inJune 2003; over 200 Y-STR loci identified (only 20 in 2000) Selection of core Y-STR loci (SWGDAM Jan 2003) Multiple commercial Y-STR kits released– Y-PLEX 6,5,12 (2001-03), PowerPlex Y (9/03), Yfiler (12/04) Many population studies performed and databasesgenerated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STRtesting along with court acceptance(From Nature website)Traits found on the Y - ChromosomeTHE HUMAN Y CHROMOSOME:AN EVOLUTIONARY MARKERCOMES OF AGEAn Early Y-Chromosome Map spittingMark A. Jobling & Chris Tyler-SmithNature Reviews Genetics (2003) 4, 598-612Abstract incessant use of TV remote buttons10,000X magnificationof X and Y chromosomes Until recently, the Y chromosome seemed to fulfill therole of juvenile delinquent among humanchromosomes — rich in junk, poor in useful attributes,reluctant to socialize with its neighbors and with aninescapable tendency to degenerate. The availabilityof the near-complete chromosome sequence, plusmany new polymorphisms, a highly resolvedphylogeny and insights into its mutation processes,now provide new avenues for investigating humanevolution. Y-chromosome research is growing up. if lost, cannot stop and ask fordirections ability to recall facts aboutbaseball/basketball/hockey/golf/etc. male pattern baldness congregates with other Ychromosome bearers to do “guythings” Source of “Testosterone poisoning”Science (1993) STpub.htm1
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressValue of Y-Chromosome MarkersDisadvantages of the Y-ChromosomeJ.M. Butler (2005) Forensic DNA Typing, 2nd Edition; Table 9.1ApplicationAdvantageForensic casework onMale-specific amplification (can avoid differentialsexual assault evidence extraction to separate sperm and epithelial cells)Paternity testingMale children can be tied to fathers in motherlesspaternity casesMissing personsinvestigationsPatrilineal male relatives may be used forreference samplesHuman migration andevolutionary studiesLack of recombination enables comparison of maleindividuals separated by large periods of timeHistorical andgenealogical researchSurnames usually retained by males; can make linkswhere paper trail is limited Loci are not independent of one another andtherefore rare random match probabilities cannot begenerated with the product rule; must use haplotypes(combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STRhaplotype (barring mutation) and thus fathers, sons,brothers, uncles, and paternal cousins cannot bedistinguished from one another Not as informative as autosomal STR resultsScenarios Where Y-STRsForensic Advantages of Y-STRs Male-specific amplification extends range of cases accessible toobtaining probative DNA results (e.g., fingernail scrapings, sexualassault without sperm) Technical simplicity due to single allele profile; can potentiallyrecover results with lower levels of male perpetrator DNA becausethere is not a concern about heterozygote allele loss via stochasticPCR amplification; number of male contributors can be determinedCan Aid Forensic Casework Sexual assaults by vasectomized or azoospermic males (nosperm left behind for differential extraction) Extending length of time after assault for recovery ofperpetrator’s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Courts have already widely accepted STR typing, instrumentation,and software for analysis (Y-STR markers just have different PCRprimers) Male-male mixtures Acceptance of statistical reports using the counting method dueto previous experience with mtDNA Gang rape situation to include or exclude potential contributors Other bodily fluid mixtures (blood-blood, skin-saliva)Some Reported Casework ExamplesY-STRs in CaseworkJ.M. Butler (2005) Forensic DNA Typing, 2nd Edition; Table 9.7Kit/Loci UsedReferenceCommentsIn-house assay withDYS19, DYS390,DYS389I/IIPrinz et al. (2001) Forensic Sci.Int. 120: 177-188In one year at the New York City Office of the Chief Medical Examiner, Y-STR testingwas performed in more than 500 cases with over 1000 evidence and referencesamples examined. A full or partial profile was obtained on 81% of all tested evidencesamples (740 worked/915 samples tested). Mixtures of at least two males wereobserved in 97 instances. In male/female mixtures of up to 1:4000, the malecomponent could be cleanly detected.In-house assay with 9 YSTR loci amplified in 3PCR reactionsDekairelle and Hoste (2001)Forensic Sci. Int. 118: 122-125Y-STR typing was attempted on 166 semen traces from 89 cases that failed to yield adetectable male autosomal profile following differential extraction. About half of thecases had sufficient DNA to produce a Y-STR profile.In-house assay withDYS393, DYS389I/IISibille et al. (2002) ForensicSci. Int. 125: 212-216Y-STR results could still be obtained more than 48 hours after the sexualassault in 30% of the cases examined. In 104 swabs collected with no evidence ofsperm, Y-STRs could be detected in 29% of the samples tested.In-house assay withDYS19, DYS390,DYS389I/IIPrinz (2003) Forensic Sci. Rev.15: 189-196Six case studies are reviewed along with advantages and disadvantages of Y-STRtesting in each case: (1) different semen donors on vaginal swab and underwear; (2)possible oligospermic perpetrator gave a nice Y-STR profile but failed to have a“male” fraction with differential extraction; (3) oral intercourse with no autosomalresults—not possible to enrich male cell fraction with differential extraction in casesinvolving saliva; (4) presence of multiple semen donors created a complex autosomalmixture that could be sorted out with Y-STR results; (5) sperm cell fraction lackedamelogenin Y-specific peak due to known deletion—Y-STR results confirmed that thesperm cell fraction DNA was of male origin; and (6) Y-STR testing was used torapidly screen 18 semen stains for comparison to 5 suspects and thus save the timeof performing the differential extractionY-PLEX 6 andY-PLEX 5 kitsSinha (2003) Forensic Sci. Rev.15: 197-201Five cases are reviewed: (1) criminal paternity case with a male fetus where thealleged father could not be excluded as the biological father; (2) autosomal STR testresulted in an uninterpretable mixture—suspect was excluded at 3 of the 7 Y-STRloci tested; (3) Y-PLEX 6 STR profile matched suspect with sweat stains on clothfound at crime scene; (4) fingernail cuttings from a victim matched a suspect at 11 YSTR loci while another suspect was excluded at 2 loci; (5) semen positive stain withno sperm cells produced a Y-PLEX 6 profile consistent with the male suspectY-PLEX 6 andY-PLEX 5 kitsSinha et al. (2004) J. ForensicSci. 49: 691-700Seven cases are reviewed (some are the same as Sinha 2003) and a list of caseswhere Y-STR results have been accepted in U.S. courts is provided.Sinha et al. (2004) J. Forensic Sci. 49: STpub.htm2
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressY-STRs can permit simplification of male DNAidentification in sexual assault casesPowerPlex Y Performance in Our Hands2 ng maleNo signal observedFemale VictimDNA Profile2 ng male: 15 ng femaleMale PerpetratorDNA Profile500 pg male: 408 ng femaleDNA Profile fromCrime SceneAutosomal STRProfile800X female DNAY-Chromosome STRProfile1 ng male: 816 ng femaleButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.2, Elsevier Science/Academic Press“Full” Y-Chromosome SequenceYpSkaletsky et al. (2003) Nature 423: 825 - 837 (19 June 2003)YqcentromereEuchromatic region(23 Mb)Y-Chromosome Sequence PublishedHeterochromatic region(not sequenced) 30 MbSequence reported in June 2003as part of Human Genome ProjectJune 19, 2003 tititisismuchmuchmoremorecomplexcomplexthanthan previouslypreviouslythought thought Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.4(B), Elsevier Science/Academic PressX- and Y-ChromosomesRecombine at Their TipsX-Chromosome Sequence PublishedRoss et al. (2005) Nature 434: 325 - 337 (17 March 2005)Xrec154 MbombinationYPseudoautosomalRegion 1 (PAR1)ationmbinreco50 MbNon-RecombiningPortion of Y Chromosome(NRY)PseudoautosomalRegion 2 (PAR2)Male-specific regionof the Y (MSY)PCR primers for Y-STRs need to be carefullydesigned to avoid X-chromosome homology(e.g., DYS391 in Y-PLEX 6 kit)Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.4(A), Elsevier Science/Academic pub.htm3
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressDYS391 Primer ImprovementsDifferent Inheritance PatternsFemale artifact problems seen with published and Y-PLEX 6 primersNew PCR primers designed at NISTLineage MarkersYCODIS STR Loci**XDeleted regions comparedto Y homolog*** * *Mutations relativeto Y homologSignificant homology exists between X and Y sequences for theDYS391 locusDYS391 Allelic LadderWe have designed primers to anneal to regions that onlyappear on the Y chromosome (targeted X deletion regions)AutosomalY-ChromosomeMitochondrial(passed on in part,from all ancestors)(passed on complete,but only by sons)(passed on complete,but only by daughters)These primers produce smaller PCR products and wereadopted by Promega for their PowerPlex Y kitButler et al. (2002) Forensic Sci. Int. 129:10-24Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.1, Elsevier Science/Academic PressY-STRs permit extension of possible referencesamples in missing persons casesCore Y-STR Lociuncle?3rd cousinButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.3, Elsevier Science/Academic PressHistory of Y STR Marker DiscoveryY STR Typing of Duplicated Regions“multi-copy loci”“Extended Haplotype”1992 - DYS19 (Roewer et al.)1994 - YCAI a/b, YCAII a/b, YCAIII a/b, DXYS156 (Mathias et al.)1996 - DYS389I/II, DYS390, DYS391, DYS392, DYS393 (Roewer et al.)1996 - DYF371, DYS425, DYS426 (Jobling et al.)1997 - DYS288, DYS388 (Kayser et al.)“Minimal Haplotype”1998 - DYS385 a/b (Schneider et al.)1999 - A7.1 (DYS460), A7.2 (DYS461), A10, C4, H4 (White et al.)2000 - DYS434, DYS435, DYS436, DYS437, DYS438, DYS439 (Ayub et al.)2000 - G09411 (DYS462), G10123 (de Knijff unpublished)U.S. Haplotype2001 - DYS441, DYS442 (Iida et al.)2002 - DYS443, DYS444, DYS445 (Iida et al.); DYS446, DYS447, DYS448,DYS449, DYS450, DYS452, DYS453, DYS454, DYS455, DYS456,DYS458, DYS459 a/b, DYS463, DYS464 a/b/c/d (Redd et al.)2002 – DYS468-DYS596 (129 new Y STRs; Manfred Kayser GDB entries)2003 – DYS597-DYS645 (50 new Y STRs; Manfred Kayser GDB entries)a repeatb repeatMultiple primer binding sites occurgiving rise to more than one PCRproduct for a given set of primersY-PLEX 6 resultsa ba bDYS385DYS385a/ba/bandandYCAIIYCAIIa/ba/bY-STR loci are often counted by thenumber of amplicons rather thanthe number of PCR primer pairsFrom J.M. Butler (2003) Recent developments in Y-STR and Y-SNP analysis. Forensic Sci. Rev. NISTpub.htm4
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressSWGDAM Sub-Committeeon the Y Chromosome Formed in July 2002 election ofofU.S.U.S. CoreCore Loci:Loci:DYS19,DYS19,DYS385DYS385 DYS439DYS43911 PCR products9 primer setsCore Y-STR CharacteristicsSTR MarkerPosition(Mb)DYS3933.17Repeat AGAT8-150.38%0.20%,0.31%Jack Ballantyne (UCF) – chairMecki Prinz (NYC) – co-chairJohn Butler (NIST)Ann Gross (MN)Jill Smerick (FBI)Sam Baechtel (FBI)John Hartmann (Orange Co.)Jonathan Newman (CFS)Phil Kinsey (OR)Gary Sims (CA DOJ)Bruce Budowle (FBI) – removedin 2004(A)R primer– All results completed agreedwith NIST results sent toparticipating labs in Dec 2002 Jack Ballantyne’s lab and JohnButler’s lab to examineadditional Y-STR and Y-SNPmarkersMulti-Copy (Duplicated) MarkerabF primerDuplicated regions are40,775 bp apart and facingaway from each other13.05[TCTG] [TCTA]DYS43813.38TTTTC6-140.09%DYS39015.71[TCTA] [TCTG]17-280.32%IIDYS385 a/b19.19,19.23GAAA7-280.23%F primerDYS39220.97TAT6-200.05%DYS389 I/II(B)a ba bSingle Region but Two PCR Products(because forward primers bind twice)IDYS389IDYS389IIF primerPositions in megabases (Mb) along the Y-chromosome were determined with NCBI build 35 (May 2004)using BLAT. Allele ranges represent the full range of alleles reported in the literature. Mutation ratessummarized from YHRD (http://www.yhrd.org; accessed 6 Apr 2005).www.cstl.nist.gov/biotech/strbase Testing of Y-PLEX 6 and YPLEX 5 kits in all labsR primerF primerDYS389 I/IIExample Y STR Fact Sheet from STRBase 60 sample set selected forscreening markers and initialtestingDYS385 a/b9-17 /24-34Butler, J.M. (2005) Genetics and genomics of core STR loci used in human identity testing. J. Forensic Sci., in press. U.S. CORE Y-STR LOCIselected in January 2003R primerButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.5, Elsevier Science/Academic PressY-Chromosome Standard NIST SRM 2395Human Y-Chromosome DNA Profiling StandardWe would like to collect variant allelesfor Y STRs as they are discovered 5 male samples 1 female sample (neg. control) 100 ng of each (50 µL at 2 ng/µL) 22 Y STR markers sequenced 9 additional Y STR markers typed 42 Y SNPs typed with Marligen kitCFBEADCertified for all loci in commercial Y-STR kits:Y-PLEX 6SWGDAMSWGDAM recommendedrecommended loci:loci:Y-PLEX 5DYS19,DYS19, DYS385DYS385 a/b,a/b, DYS389I/II,DYS389I/II,Y-PLEX 12DYS390,DYS391,DYS390, DYS391, DYS392,DYS392,DYS393, DYS438,DYS438, DYS439DYS439PowerPlex Y DYS393,Y-filer - adds DYS635 (C4); now sequencedHelps meet FBI Standard 9.5 (and ISO 17025) traceability to a national ISTpub.htm5
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressSequence Determination of Y STR Repeat Regionfor Each ComponentDYS392 (forward) AY STR Allele NomenclaturesSRM 2395 as an international standard should help resolve issues13 TAT repeatsDYS19DYS392 (forward) BDYS392 (forward) CDYS392 (forward) DDYS392 (forward) E11 TAT repeats11 TAT repeats3[TAGA]Sequencing PerformedDYS19DYS385 a/bDYS389 48Y-GATA-H4DYS460 (A7.1)DYS461 (A7.2)DYS46211 TAT AGG [TAGA]10-19 totalDYS4393 different nomenclatures have been published for DYS4392[GATA]9-14 variable311[GATA][GATA][GATA]N412 TAT repeatsN14Nowaccepted wayN3[GATA]N716-21 totalCommercial Y-STR Kits(Minimal/extendedhaplotype)(Redd et al.)DYS446DYS447A7.2 Bosch et al.)43 (51) Y-STRs DYS456G09411 (DYS462)DYS458(217 with Manfred’s)DYS459 a/bY-PLEX 6 (ReliaGene)DYS463Y-PLEX 5 (ReliaGene)DYS464 a/b/c/dDYS389I/IIDYS390Available Y-STR Kits(Iida et al.)(Ayub et al.)(White et al.)A7.1 (DYS460)DYS19DYS391DYS392DYS393DYS385 a/bYCAII a/bDYS388DYS425DYS434DYS441DYS435DYS442Y-PLEX 12 (ReliaGene)PowerPlex Y (Promega)Yfiler (Applied Biosystems)DYS426YCAIII a/bDYS468-DYS645166 new Y STRs(Manfred(Manfred KayserKayser GDBGDB entries)entries)Allele size range and locus dye colorsNIST 20plex200 bp100 bpDYS391DYS389I426DYS393The Manly-plex460DYS437YCAIIa/bH4300 bpDYS439DYS390388Published Sept 2002DYS19447DYS389II400 bpDYS438DYS385a/bDYS392448PowerPlex YReleased by Promega Corporation in Sept 2003Relative Fluorescence UnitsPCR Product Size (bp)200 bp100 bpY STR 20plex Amplification391426 460439393 YCAII390 447 19a/b 389IH4388 437385a/bDYS391389II rbase/NISTpub.htmDYS393300 bpDYS439DYS389IDYS437DYS19DYS390400 bpDYS389IIDYS392A few minor primermodifications withsome lociDYS385a/b6
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressPromega PowerPlex Y Allelic LaddersDYS391DYS389IDYS439PowerPlex Y Validation StudiesDYS389IIForensic Science International 148 (2005) 1-14DYS438DYS437DYS393DYS19DYS390DYS392DYS385 a/bU.S. Core Loci DYS437Single amplification; ladders contain 103 alleles# RunDescription of Samples Tested (performed in 7 labs and Promega)5 samples x 8 labs40Mixture Ratio (male:female)6 labs x 2 M/F mixture series x 11 ratios(1:0,1:1,1:10,1:100,1:300,1:1000,0.5:300, 0.25:300,0.125:300,0.0625:300, 0.03:300 ng M:F )132Mixture Ratio (male:male)6 labs x 2 M/M mixtures series x 11 ratios (1:0, 19:1, 9:1, 5:1, 2:1, 1:1,1:2, 1:5, 1:9, 1:19, 0:1)132Sensitivity7 labs x 2 series x 6 amounts (1/0.5/0.25/0.125/0.06/0.03)84Non-Human24 animals24NIST SRM6 components of SRM 23956Precision (ABI 3100 and ABI 377)10 ladder replicates 10 sample replicated [8 ladders 8 samplesfor 377]36Non-Probative Cases65 cases with 102 samples102Stutter412 males used412Peak Height RatioN/A (except for DYS385 but no studies were noted)Cycling Parameters5 cycles (28/27/26/25/24) x 8 punch sizes x 2 samples80Annealing Temperature5 labs x 5 temperatures (54/58/60/62/64) x 1 sample25Reaction volume5 volumes (50/25/15/12.5/6.25) x [5 amounts 5 concentrations]50Thermal cycler test4 models (480/2400/9600/9700) x 1 sample [3 models x 3 sets x 12 samples]76Male-specificity2 females x 1 titration series (0-500 ng female DNA) x 5 amounts each10TaqGold polymerase titration5 amounts (1.38/2.06/2.75/3.44/4.13 U) x 4 quantities (1/0.5/0.25/0.13 ng DNA)20Primer pair titration5 amounts (0.5x/0.75x/1x/1.5x/2x) x 4 quantities (1/0.5/0.25/0.13 ng DNA)20Magnesium titration5 amounts (1/1.25/1.5/1.75/2 mM Mg) x 4 quantities (1/0.5/0.25/0.13 ng DNA)Krenke et al. (2005) Forensic Sci. Int. 148:1-14TOTAL SAMPLES EXAMINEDNIST Multiplexes for High-Throughput Y STR TypingY-STR KitLaid the Groundwork for Applied Biosystems’ Yfiler11 U.S. core lociPCR Product Size (bp) DYS437 DYS448393390 DYS456385 a/b DYS458391YCAII a/b DYS635426439H4460 H4388Relative Fluorescence UnitsValidation Summary Sheet for PowerPlex YStudy Completed (17 studies done)Single Source (Concordance)43720plex389II389I44744819392385 a/b44745645045843811plex464 a/b/c/d448201269Schoske et al. (2004) High-throughput Y-STR typing of U.S. populations , Forensic Sci. Int., 139:107-121Yfiler Allelic LaddersAmpFlSTR Yfiler Released by Applied Biosystems in Dec 2004200 bp100 bp6-FAMDYS456DYS389I300 bpDYS390400 S448RedLIZOrangeGS500-internal size ISTpub.htm7
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressOur Group at NIST Was Involved inBeta-Testing All U.S. Based Y-STR KitsCommercial Y-STR Kits Available ReliaGene Technologies (New Orleans, LA)– Y-PLEX 6: DYS19, DYS389II, DYS390, DYS391, DYS393, DYS385 a/b– Y-PLEX 5: DYS389I/II, DYS392, DYS438, DYS439– Y-PLEX 12: DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391,DYS392, DYS393, DYS438, DYS439, amelogeninNIST SRM 2395 Component AY-PLEX 12 Promega Corporation (Madison, WI)– PowerPlex Y: DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391,DYS392, DYS393, DYS438, DYS439, DYS437 Applied Biosystems (Foster City, CA)PowerPlex Y– Yfiler : DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391, DYS392,DYS393, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458,DYS635 (Y-GATA-C4), Y-GATA-H4 Serac (Bad Homburg, Germany)DYS438 DYS393DYS391DYS389IDYS437DYS439DYS389IIDYS390 DYS19 DYS385DYS392Yfiler– genRES DYSplex-1: DYS389I/II, DYS390, DYS391, DYS385 a/b,amelogenin– genRES DYSplex-2: DYS19, DYS389I/II, DYS392, DYS393 Biotype (Dresden, Germany)– Mentype Argus Y-MH: DYS19, DYS385 a/b, DYS389I/II, DYS390,DYS391, DYS392, DYS393New Y-Chromosome InformationResources on STRBaseAvailable Y-STRHaplotype DatabasesLargest Y-STRDatabaseY-Chromosome Haplotype ReferenceDatabase (YHRD)Run only with minimal haplotypehttp://www.yhrd.orgAs of 12/17/04:28,650 haplotypes6,281 haplotypeswith all US required lociCommercial Y-STR kits exist to amplifyall of the core loci in a single reaction(plus a few additional 385 a/bUS haplotype requires2 additional strbase/NISTpub.htmYfiler Haplotype abase/#Samples Contributed by NIST259 African Americans3 Asians262 Caucasians139 HispanicsData provided by NIST663/3561 18.6%8
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressPowerPlex Y Haplotype xy/595 Caucasians284 Asians630 Hispanics577 African Americans357 Native Americans--------------1311 Caucasians325 Asians894 Hispanics1108 African Americans366 Native Americans--------------2,443 total4,004 totalY-STR Population StudiesMarch 2005PowerPlex Y Population StudyA few recent Y-STR population studiesPopulation5 North AmericangroupsU.S. Caucasians,African Americans,HispanicsAustrian91 European groups# Samples# Loci2,4431264722(27)1351712,7007ReferenceBudowle et al. (2005) FSI 150:1-15Schoske et al. (2004) FSI 139:107-121Berger et al. (2005) IJLM, in press (Yfiler)Roewer et al. (2005) Hum Genet 116:279-291More than 200 Y-STR population studies have beenpublished (most of this data is deposited in the YHRD– Y Chromosome Haplotype Reference Database)Initial 2,443 Samples inPowerPlex Y Haplotype DatabaseCompilation of Y STR Population DataAppendices in Rich Schoske’s Ph.D. dissertation; available on STRBase159 Canadian samplesPowerPlex Y Haplotype xy/ Source: over 200 published population data papers Helps define observed allele ranges, which aids in multiplex assaydevelopment (spacing between loci in the same dye color) Information is available to the community through the STRBase website– permits analysis of optimal markers for particular /NISTpub.htm9
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressRichard Schoske se/pub pres/Schoske2003dis.pdf Worked at NIST from Nov2000 to May 2003 270 page Ph.D. dissertationdGra,y 11Madeuat320 0 Entitled “The design,optimization and testing of Ychromosome short tandemrepeat megaplexes.” Available for download onNIST STRBase websiteRich SchoskePhD student from American UniversityFunded by Air ForceStandard U.S. Population STpop.htm260 Caucasians, 260 African Americans, 140 Hispanics, 3 Asians 663DNA extracted from whole blood (anonymous;self-identified ethnicities) received fromInterstate Blood Bank (Memphis, TN) andMillennium Biotech Inc. (Ft. Lauderdale, FL)malesextracted genomic DNAStock tubesTo date: ( 95,000 allele calls)Identifiler (15 autosomal markers Amelogenin) (10,608)Roche Linear Arrays (HV1/HV2 10 regions) (6,630)Y STRs 22 loci—27 amplicons (17,388)Y STRs 27 new loci (14,535)Yfiler kit 17 loci (11,237)Y SNPs 50 markers on sub-set of samples (11,498)Orchid 70 autosomal SNPs on sub-set (13,230)miniSTR testing-new loci and CODIS concordance (9,228)mtDNA full control region sequences by AFDILGenotypes withvarious humanidentity testingmarkersNIST Multiplexes for High-Throughput Y STR TypingU.S. Population Data on 22 Y-STRs22 loci27 PCR productsPCR Product Size (bp)393390391Relative Fluorescence Units42646020plex385 a/bYCAII a/b439H4388437389II389I4474481939243817,388 allele calls with 2 multiplexes and385 a/bhours on ABI 3100)14 96-well plates ( 10044711plex456450464 a/b/c/d458448pdf file available at tmSchoske et al. (2004) High-throughput Y-STR typing of U.S. populations , Forensic Sci. Int., 139:107-121US haplotype(Reliagene kits)Yfiler(ABI)PowerPlex Y(Promega) C4Statistical Calculations on Y-STR Data Locus (gene) Diversity (n/n-1)(1 - Σpi2) where n is the numberof samples in the dataset and pi is the frequency of the ith allele Haplotype Diversity (HD) (n/n-1)(1 - Σpi2) where n is thenumber of samples in the dataset and pi is the frequency of the ithhaplotype Random Match Probability (RMP) 1 - HD Discrimination Capacity (DC) – total number of observed haplotypesdivided by the total number of individuals in the dataset Unique Haplotypes (UH) – number of haplotypes that occur only oncein the datasetSchoske et al. (2004) High-throughput Y-STR typing of U.S. populations , Forensic Sci. Int., e/NISTpub.htm10
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressY-STR MarkerCombinations260 African Americans244 Caucasians143 HispanicsHDRMPHDRMPHDY-PLEX 6 al” tended” S. STR 11plex0.99930.00070.99870.00130.99920.0008Y-STR 20plex0.99980.00020.99980.00020.99980.000222 Y-STRs0.99990.00010.99990.00010.99990.0001Top 10 (w/o YCAII a/b)0.99990.00010.99990.00010.99990.0001Y-STR MarkerCombinations260 African AmericansDCUHDCUHDCY-PLEX 6 kit82.3%18868.9%13678.3%97“minimal” haplotype88.5%21375.8%16181.1%100“extended” haplotype91.9%22783.6%18489.5%120“U.S. haplotype”91.9%22282.3%17693.3%121Y-STR 11plex93.1%22788.5%19894.4%127Y-STR 20plex98.5%25297.2%23098.6%13922 Y-STR Markers98.9%25499.6%24299.3%141Top 10 (w/o YCAII a/b)96.9%24497.5%23299.3%141244 CaucasiansStatistics with Y-STRHaplotypes143 HispanicsUHMost labs will probably go with thecounting method (number of times ahaplotype is observed in a database)as is typically done with mtDNA resultsSchoske et al. (2004) High-throughput Y-STR typing of U.S. populations , Forensic Sci. Int., 139:107-121Y-Chromosome Haplotype Reference Databasewww.YHRD.orgExample Y-STR Haplotype7 matches in 27,773individuals from 236worldwide populationsRelease "15" from 2004-12-17 16:11:24Core US Haplotype Matches by DatabasesDYS19 – 14DYS389I – 13DYS389II – 29DYS390 – 24DYS391 – 11DYS392 – 14DYS393 – 13DYS385 a/b – 11,15DYS438 – 12DYS439 – 13Minimal Haplotype ResultDYS19 – 14DYS389I – 13DYS389II – 29DYS390 – 24DYS391 – 11DYS392 – 14DYS393 – 13DYS385 a/b – 11,15 YHRD (9 loci)– 7 matches in 27,773 YHRD (11 loci)– 0 matches in 6,281 ReliaGene (11 loci)– 0 matches in 3,403 PowerPlex Y (12 loci)– 0 matches in 4,004 Yfiler (17 loci)– 0 matches in 3,561Frequency Estimate CalculationsIn cases where a Y-STR profile is observed a particular number of times(X) in a database containing N profiles, its frequency (p) can becalculated as follows:p X/NWhen there is no match In cases where the profile has not been observed in a database,the upper bound on the confidence interval is7 matches in 27,7731-α1/Np 7/27,773 0.000252 0.025%An upper bound confidence interval can be placed on the profile’sfrequency using:p 1.96( p)(1 p)N0.000252 1.960 matches in 4,004where α is the confidence coefficient (0.05 for a 95% confidenceinterval) and N is the number of individuals in the database.(0.000252)(1 0.000252)27,7731-α1/N 1-(0.05)[1/4,004] 0.000748 0.075% ( 1 in 1340) 0.000252 0.000187 0.000439 0.044%( 1 in pub.htmIf using database of 2,443, then the best you can do is 1in 81611
June 8, 2005Canadian Forensic DNA Technology WorkshopJ.M. Butler, “State of the Y Chromosome” addressDifficult Questions The Meaning of a Y-Chromosome MatchConservative statement for a match report:The Y-STR profile of the crime sample matchesthe Y-STR profiles of the suspect (at xxxnumber of loci examined). Therefore, wecannot exclude the suspect as being thedonor of the crime sample. In addition, wecannot exclude all patrilineal related malerelatives and an unknown number ofunrelated males as being the donor of thecrime sample. Which database(s) should be used for Y-STR profilefrequency estimate determination? Are any of the current forensic Y-STR databasestruly adequate for reliable estimations of Y-STRhaplotype frequencies?– Some individuals share identical Y-STR hap
DNA Profile Male Perpetrator DNA Profile DNA Profile from Crime Scene No signal observed Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.2, Elsevier Science/Academic Press Y-STRs can permit simplification of male DNA identification in sexual assault cases 2 ng male 2 ng male: 15 ng female 500 pg male: 408 ng female 1 ng male .
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Chromatin is divided into euchromatinand heterochromatin The sister chromatids of a mitotic pair each consist of a fiber ( 30 nm in diameter) compactly folded into the chromosome 15 Chromatin is divided into euchromatinand heterochromatin
Telomere position effect (TPE) is one heterochromatic silencing phenomenon. TPE refers to the silencing of genes near telomeres and is classically defined as a continuous spread of heterochromatin from the telomere inward, although discontinuous heterochromatin formation has also been observed at telomeres in different organisms. TPE has been
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