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McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs,-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5& Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR Kits PowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)x ddd  o   Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)\dd  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> # Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate, K *Duplications and Deletions of Y-Chromosome++Y-STR Mixtures8Statistical treatment is not the same as autosomal STRs ,) ~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples(( #Differences from Reference SequenceChallenges with mtDNAsData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes DNA Database Quality66*6* T'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88  &2A Common Use of mtDNA Analysis is for Hair ShaftsRPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSf/      0` 33` Sf3f` 33g` f` www3PP` ZXdbmo` \ғ3y`Ӣ` 3f3ff` 3f3FKf` hk]wwwfܹ` ff>>\`Y{ff` R>&- {p_/̴>?" dd@$|?" dd@  " @ ` n?" dd@   @@``PR    @ ` ` p>> f(    6v   ` v  T Click to edit Master title style! !  0t+j   ` v  RClick to edit Master text styles Second level Third level Fourth level Fifth level!     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( .`   HB /` C D@ @ z    0`# S"  ( 1`   HB 2` C D@ @ z    3`# S" ?7B 4` 3   HB 5` C D@ @ z    6`# S" 7< 7` #   HB 8` C D@ @ z    9`# S"z  ( :`   HB ;` C D@ @ z    <`# S"z 7< =` #   HB >` C D@ @ VB ?` C D"  VB @` C D" 4 VB A` C D"  z    B`# S"B T ( C`   HB D` C D@ @ z    E`# S"5 B u ( F`   HB G` C D@ @ z    H`# S" 5 u ( I`   HB J` C D@ @ z    K`# S" u ( L`   HB M` C D@ @ z    N`# S"u B ( O`   HB P` C D@ @ VB Q` C D"  5 VB R` C D"5 6 5 z    S`# S" < T` #   HB U` C D@ @ z    V`# S" B < W` #   HB X` C D@ @ VB Y` C D" s VB Z` C D"2VB [` C D"RRVB \` C D"RVB ]` C D"RVB ^` C D"  R _` 3 7" c K Eston Hemings" bB `` c $DԔ"cz    a`# S"[T( b`   HB c` C D@ @ z    d`# S"T"( e`   HB f` C D@ @ z    g`# S"" ( h`   HB i` C D@ @ z    j`# S"[ V < k` #   HB l` C D@ @ z    m`# S"[  ( n`   HB o` C D@ @ z    p`# S"[ ( q`   HB r` C D@ @  s` 3 D"c, LThomas Woodson"Bz    t`# S"V " x u` S HA*Wide upward diagonal  B v` ZDA*Wide upward diagonal@ @ z    w`# S" "V ( x`   HB y` C D@ @ z    z`# S" " ( {`   HB |` C D@ @  }` C "7] QDifferent Y Haplotype B ~` C ĸ" V D  QSame Y Haplotype$B ` C " VJefferson Y Haplotype$B ` C ¨"G +  VJefferson Y Haplotype$B ` 3 _"D   E?(F; ` <g' _Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic PressT`     ( X ` C 0A TJeffersonf ` 6t +N 1Historical Investigation of Jefferson-Hemings DNA22&z T 4  `  ,$D  0 ` 0TzT 4  20T  0  `#   _ ` c <A ftdna_logo 0 7   0 ` C bA 8OAlogo10Oxford Ancestors large logo7 @  ` c RA  $logoRelative Genetics J  0b ` C 2Aheader_logo  ` <$~ w q  SGenetic Genealogy Companies0  ` <܆6Q r  -SOURCE: Foster et al. (1998) Nature 396:27-28T. H ` 0޽h ? 33___PPT10.Ģ+=kD~' = @B D9' = @BA?%,( < +O%,( < +Dp' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*`%(D' =+4 8?RCBBCB#ppt_wB*Y3>B ppt_w<*`D' =+4 8?RCBBCB#ppt_hB*Y3>B ppt_h<*`D' =+4 8?RCB?BCB#ppt_xB*Y3>B ppt_x<*`D' =+4 8?RCB?BCB#ppt_yB*Y3>B ppt_y<*`+  0L0 <=(  <x < c $X   ]L +# <# |z < 6+ U Application. 0 bB0 < 6䓨 S Advantage. 0 bB0 < 6+& Z <00 !$ < 6p& Z <00 !$ < 6+&, z,Forensic casework on sexual assault evidence2-0,dfF$f,  < 6২&, fMale-specific amplification (can avoid differential extraction to separate sperm and epithelial cells)<g0dfK`B$  < 6ܝ+, Z <00 !$  < 6C, Z <00 `B$  < 6Ȩ+ WPaternity testing*0`B$  < 6dͨ BMale children can be tied to fathers in motherless paternity cases*C0B`B$ < 6(Ҩ+X  Z <00 !$ < 6רX  Z <00 !$ < 6ݨ+X ^  dMissing persons investigations*0`B$ < 6xX ^  <Patrilineal male relatives may be used for reference samples4=0<dF$ < 6,+^   Z <00 !$ < 6 ^   Z <00 !$ < 6,+  n(Human migration and evolutionary studies*)0(`B$ < 6lӕ  _Lack of recombination enables comparison of male individuals separated by large periods of time*`0_`B$ < 6ؕ+ Z <00 !$ < 6|ݕ Z <00 !$ < 6+ j$Historical and genealogical research*%0$`B$ < 60 OSurnames usually retained by males; can make links where paper trail is limited*P0O`B$ < 6+# Z <00 !$ < 6ӕ# Z <00 !$  < <<9 >J.M. Butler (2005) Forensic DNA Typing, 2nd Edition; Table 9.1Z?0H < 0޽h ? ̙33y___PPT10Y+D='  = @B +k  0 z4(  4l 4 S 6A?423810a-f1"`@x 4 c $H     x 4 c $  P   4 0(6P g-10,000X magnification of X and Y chromosomes.0. 4 < 4 w(From Nature website)F0  4 <&~  @Abstract 0 H 4 0޽h ? ̙33___PPT10i.e`+D='  = @B + 0   8w (  8L 8 c $R(A28@  8 0-`. 8 spitting incessant use of TV remote buttons if lost, cannot stop and ask for directions ability to recall facts about baseball/basketball/hockey/golf/etc. male pattern baldness congregates with other Y-chromosome bearers to do  guy things Source of  Testosterone poisoning .1 2 8 00- j"Traits found on the Y - Chromosome,#0 2" pB 8@ HD?PPp2 8 H Ԕ?p pB 8@ HDԔ?P@ pB 8@ HDԔ?@ppB  8@ HD?PppB  8@ HDԔ? @pB  8 HD? 0 pB  8 HDԔ? 0 pB  8 HDԔ?` @p pB 8 HDԔ?  pB 8 HDԔ? @z  I\ 8 I`,$D 0N    8 ` 0@ 8 <A ??    8 <>@ TScience (1993) 261:679"0 8 <`D I\0 SAn Early Y-Chromosome Map0fH 8 0޽h ? ̙33~___PPT10^+31DB'  = @B D' = @BA?%,( < +O%,( < +D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*8%(+  0 00(  0x 0 c $ J`p   x 0 c $J 0   H 0 0޽h ? ̙33___PPT10i.f*+D='  = @B +0   0 @b(  @~ @ s *TW `    @ c $@X<$ 0  H @ 0޽h ? ̙33  ___PPT10 .aP+[W_Dz '  = @B D5 ' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*@%(DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*@y%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*@z%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*@%(+8+0+@0 +   0 Db(  D~ D s *@p `    D c $q <$ 0  H D 0޽h ? ̙33  ___PPT10 ..b%+[W_D* '  = @B D ' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*D%(DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*D%(DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*D4%(DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*D5%(+8+0+D0 +  0  H0(  Hx H c $䀕   x H c $  H H 0޽h ? ̙33___PPT10i.fp7+D='  = @B + 0   PT (  T T 0S  HAutosomal STR Profile T 0 =  JY-Chromosome STR Profile F M  T M vN _* T MC ZR T s *ZR T s *_TB T c $D*_*vN -_  T wC ZR  T s *-ZR  T s *_-TB  T c $D_ZR  T s * u ZR T s *C u TB T c $D C ZR T s *k u ZR T s *C u  T 0x+ -Female Victim DNA Profile T 0<7  0Male Perpetrator DNA Profile T 0P W  0DNA Profile from Crime SceneZR T s *- i TB T c $D6t t ZR T s *-e TB T c $D6  TB T c $D6 T 0谕+ FNo signal observedc T 0 P cButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.2, Elsevier Science/Academic Press d0       &   T 6 `00 SY-STRs can permit simplification of male DNA identification in sexual assault casesTTH T 0޽h ? 33___PPT10i.C +D='  = @B +@  0L0 OG`(  `x ` <A ??`   ` <HÕGvA ? 2 ng male 0  ` <Ǖ F M2 ng male: 15 ng female0 ` <L̕ F w  P500 pg male: 408 ng female0 ` <͕W v Q N1 ng male: 816 ng female0x ` c $ѕ*    ` 6k J e G800X female DNA0H ` 0޽h ? ̙33y___PPT10Y+D='  = @B +  0 pU(  x  <A  ??  #  6 ?f i> mSelection of U.S. Core Loci: DYS19, DYS385 a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS4394n0 Rr  S  E `8   H  0޽h ? ̙33___PPT10i.ev+D='  = @B +&=  0 28*8EE7(  x  c $\ `p   2 H # #":. gggp     B ?OH c0.05%,(bB @`  B' ?OH b6-20,(bB @`  B1 ?OH aTAT,(bB @`  B; ?OH c20.97,(bB @`  BF ?OH fDYS392.(bB @`   Bd@ ? O c0.23%,(bB @`   BY ? O b7-28,(bB @`   Bc ? O bGAAA,(bB @`   B] ? O j 19.19, 19.23, ( bB @`   Bw ? O r DYS385 a/b6 ( f3F3 @`  B ?   c0.32%,(bB @`  B| ?   c17-28,(bB @`  B ?   k [TCTA] [TCTG],( bB @`  B8 ?   c15.71,(bB @`  B ?   fDYS390.(bB @`  B ?   c0.09%,(bB @`  B ?   b6-14,(bB @`  B` ?   cTTTTC,(bB @`  B ?   c13.38,(bB @`  B ?   fDYS438.(bB @`  B ?7   j 0.20%, 0.31%, ( bB @`  BĚ?7   k 9-17 / 24-34,( bB @`  B ?7   k [TCTG] [TCTA],( bB @`  B ?7   c13.05,(bB @`  B?7   s DYS389 I/II6 ( f3F3 @`  B`?>7  c0.38%,(bB @`  BX?>7  b8-15,(bB @`  Bb?>7  bAGAT,(bB @`   Bl?>7  c12.95,(bB @` ! Bhv?>7  fDYS439.(bB @` " Bp?E> c0.40%,(bB @` # B ?E> b6-14,(bB @` $ B?E> bTCTA,(bB @` % Bh?E> c12.54,(bB @` & B)?E> fDYS391.(bB @` ' B 4?LE c0.20%,(bB @` ( B.?LE c10-19,(bB @` ) BG?LE bTAGA,(bB @` * B?LE c10.12,(bB @` + BXL?LE eDYS19.(bB @` , B?SL c0.05%,(bB @` - B?SL b8-17,(bB @` . B?SL bAGAT,(bB @` / B?SL b3.17,(bB @` 0 B$g?SL fDYS393.(bB @` 1 Bg?S u Mutation Rate6( fF @` 2 Bg?S t Allele Range6 ( fF @` 3 Bg?S t Repeat Motif6 ( fF @` 4 B g?S u Position (Mb)6( fF @` 5 Bg?S r STR Marker6 ( fF @`HB 6 C ZB 7 s *1 ?HHHB 8 C HHB 9 C HZB : s *1 ?SSZB ; s *1 ?LLZB < s *1 ?EEZB = s *1 ?>>ZB > s *1 ?7 7 ZB ? s *1 ?  ZB @ s *1 ?  ZB A s *1 ?  ZB B s *1 ?OO C 0g  q Positions in megabases (Mb) along the Y-chromosome were determined with NCBI build 35 (May 2004) using BLAT. Allele ranges represent the full range of alleles reported in the literature. Mutation rates summarized from YHRD (http://www.yhrd.org; accessed 6 Apr 2005).  0  0N D <gT vButler, J.M. (2006) Genetics and genomics of core STR loci used in human identity testing. J. Forensic Sci., in press..w0[  g  E 6!g`,$D 0 m11 PCR products 9 primer sets2 0H  0޽h ? ̙33___PPT10t.a+:+҇D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(DR' =A@BB BB0B%(D' =1:Bvisible*o3>+B#style.visibility<*E%(D' =-6B%slide(fromBottom)*<3<*E+8+0+E0 +O" 0 f!^!== (    C h  J DYS385 a/b$  BF      BPCDEF̙Ԕ@P @   BC@DE F(̙Ԕ@@@@0@`@@@]  0 ̙ Ca = b"F  0 ̙5R j a bDFF  C  ^y  K DYS389 I/II$  B  <  5(A)  <ġ B  5(B)O F 2z >   z > N X 8   2z >T tB   #  -l  <oB   <Y t j ;I0T  B0  # 4 l  <o , B0   <f    <II0ZB  s *DoX 8 `  0(  `  0  `  0  `  0  `  0 $  `  0$  `  0  `  0  `  0  `B  0Do@  `B B 0Do x ZB  s *D)@  ZB   s *D)  v  ZB ! s *D)   `B " 0Do  # <( J ]  8F primer  $ < .  8F primer  % <l  8R primer F   &  N `b  ' n{ NB ( S DjJb < )B # 9TB *B c $Do``ZB + s *DoN , C @,BzjR =a @< - # < r 9TB . c $Do ` `ZB /B s *Do   0 C |40 | T =b @ 1 C 1)  FDuplicated regions are 40,775 bp apart and facing away from each other"GF 2 <Hl 8F primer  3 <zP 8R primer  4 <  x 8F primer  5 <  D 8R primer 9F   S 6  S 7  BPCDEF̙Ԕ@P @  8  BPCDEF̙Ԕ@P @ _ 9 0`c    7DYS389I : 0tO S  8DYS389II  c ; 0 P cButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.5, Elsevier Science/Academic Press d0       &   < <)g)l 0 XMulti-Copy (Duplicated) Marker03 = 0 *g p   GSingle Region but Two PCR Products (because forward primers bind twice)*H0#3%3H  0޽h ? 33___PPT10i.p 0+D='  = @B +2  0L0 110VT1(    <g&& \&Allele size range and locus dye colors'0'L L  K# wEfB L 6DԔ fB M 6D>fB N 6D>fB O 6D>fB P 6D> Q 0_ B100 bp 0( 2 R 08_Fm B400 bp 0( 2 S 0_B m B300 bp 0( 2 T 0T_& m B200 bp 0( 2T bH U# fB V 6DoHfB W 6Dobb<fB X 6DoBT bH Y# 6  fB Z 6DoHfB [ 6Dobb<fB \ 6DoBT bH ]# :fB ^ 6DoHfB _ 6Dobb<fB ` 6DoB a 0 _aDH BDYS391 0( 2 b 6_o r PowerPlex YJ 0( 2  .   c 08_aH CDYS389I 0( 2 d 00_X{  ? BDYS439 0( 2 e 0_a H DDYS389II 0( 2  f 6T!_3ԔC"?q BDYS438 0( 2 g 0l%_1  BDYS437 0( 2 h 0h)_h  q ADYS19 0( 2 i 0X-_ |q BDYS392 0( 2 j 0,1_N BDYS393 0( 2 k 0d4_  BDYS390 0( 2 l 07_N > E DYS385a/b 0( 2  m B;_"< h,Released by Promega Corporation in Sept 2003 -0-* o 0\@_  "AmpFlSTR Yfiler"!0( 2   .   &   p s *\H_? o BDYS437 0( 2 q s *LL_ o BDYS448 0( 2 r s * P_Co >H4 0( 2F F  ~ ] w fB  6DԔ fB  6D>fB  6D>fB  6D>fB  6D>  0T_ B100 bp 0( 2  0X_Fm B400 bp 0( 2  0\_B m B300 bp 0( 2  0`_& m B200 bp 0( 2T bH # fB  6DoHfB  6Dobb<fB  6DoBT bH # 6  fB  6DoHfB  6Dobb<fB  6DoBT bH # :fB  6DoHfB  6Dobb<fB  6DoB  s *lg_   BDYS456 0( 2  s *`k_   CDYS389I 0( 2  s * o_ !  BDYS390 0( 2  s *s_ o   DDYS389II 0( 2   s *v_+ e BDYS458 0( 2  s *z_+   ADYS19 0( 2  s *lq_+  E DYS385a/b 0( 2   s *,_^EE BDYS393 0( 2  s *_^< E BDYS439 0( 2  s *_^#E BDYS392 0( 2  s *T_ l BDYS438 0( 2  s *_dHK BDYS391 0( 2  s *,_f] M BDYS635 0( 2  <_ <ip  f*Released by Applied Biosystems in Dec 2004 +0+  <p_; N3 dye colors 12-plex PCR  <L_ (` N4 dye colors 17-plex PCR  <_T]; 6FL  <_% 7JOE  <t_ 3TMR  <x_ Fr  96-FAM  <x_.  7VIC  <_f.M 3NED  <_|"c 7PETH  0޽h ? ̙33y___PPT10Y+D='  = @B +I 0   @L (  x  <A( ??   <_Pq$ DDYS391"0  <_PC$ EDYS389I"0  <_PA $ DDYS439"0  <H_Pp$ FDYS389II" 0   < _/ DDYS438"033  <|__  DDYS437"033  <_  CDYS19"033  <@ DDYS392"033  < !pt  @DYS3930  <X ?  @DYS3900  < ,  D DYS385 a/b 0   < . i1Single amplification; ladders contain 103 alleles202  6  $Promega PowerPlex Y Allelic LaddersD%0.z {  ,$D 0f  6fԔ   68[{ aU.S. Core Loci + DYS437.0f fH  0޽h ? ̙335-___PPT10 +$D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BB BB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bdissolve*<3<*+A  0 $(  x  <A- ??Mp x  <A. ??U,  x  <A/ ??$ ]$  x  <A0 ??> :G   <(&   fYfiler Allelic Ladders0l . .,$D 0l  <Ԕ.1   0 @U.S. Core Loci + DYS437, DYS448, DYS456, DYS458, DYS635, GATA H48A0ff0ff   6Ԕ f   6Ԕ Y f   6Ԕ f   6Ԕ<f  6Ԕ s H  0޽h ? ̙33IA___PPT10!+n2D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(DG' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-6B'checkerboard(down)*<3<*+2 0 11P ZZD1(    s *(| 0 g#SWGDAM recommended loci (+ 438,439)($0   <d  8(26)0  s *8 0& =Minimal haplotype (19, 389I/II, 390, 391, 392, 393, 385 a/b)(>0-RB @ s *DP `RB  s *D `RB  s *D& + 0  <  7(7)0   <\ \  8(15)0   <p 7(4)0   s *̙P `7 p.PowerPlex Y (+437)&/0(RB  @ s *D RB   s *D& RB  s *D&W   <\p  7(2)0  <H  7(4)0  <4  7(1)0^  6p@ RB @ s *D RB  s *Dp   <$  7(1)0  <@  7(3)0^  60 RB @ s *D - RB  s *D- `  < 5  7(3)0  <Զ *  8(12)0  <H Os  7(1)0  <4  7  7(1)0  < ’  7(1)0F l  lf   6fԔl ! <ƒ7uW JU.S. Haplotype 0f " <˒F c'Subdividing Common Types with More Loci (0( # BLВf3?{h Y!657 males from 3 U.S. populations"0" $ <Β p  7(1)0 % <tؒ } p  7(1)0 & < ܒ A e p  7(1)0 ' <+ q  7(1)0 ( < q  7(1)0 ) < ~q  7(1)0 * < Bfq  =(3)0 + <  *q  7(1)0 , < q  7(1)0 - <t "  7(1)0 . <`   7(1)0 / << h  7(1)0 0 <$ג ,P  7(1)0 1 <j   7(1)0 2 <HB   7(1)0 3 s *p.f 5u  h&Yfiler (+448,456,458,635,H4)&'0RB 4 s *D *y RB 5@ s *D ( RB 6@ s *D ! RB 7 s *D &. F  J  8  J ZB 9B s *D  ZB : s *D J  ; < h  7(1)0 < < ,P  7(1)0F  J  =   ZB >B s *D  ZB ? s *D J  @ <fԔ 6Most common type DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  13 DYS393  13 DYS385 a/b  11,14&{0 j  A < %  7(1)0 B < Bf  7(1)0 C <  *  7(1)0 D <x   7(1)0^ E 6<   F <dfԔo u fIdentical: DYS& 444,446,485,495,505,508,534,540,556 Subdivide into two groups (2)(1): DYS& 449,463,520,532,533,557,570,594,643 Subdivide into three groups (1)(1)(1): DYS522, DYS576RB G@ s *DfjJ` _ RB H s *DfjJe j RB I s *D ` RB J s *D  RB K s *D  RB L s *D  RB M@ s *D 4 RB N@ s *D {  RB O@ s *D  RB P@ s *D   RB Q@ s *D \  RB R@ s *D  RB S@ s *D e RB T s *D ^ RB U s *D hY F  X  V  X N  J  W  X ZB XB s *D  ZB Y s *D J ZB ZB s *D H  0޽h ? ̙33y___PPT10Y+D='  = @B +  0L0 0  |(   ~   BA ?Ԕ? ~   BA5 ??<P 1 x   c $,`@   XB   0D5>    0./   LLargest Y-STR Database0   0L3) d.Locus boxes are hyperlinked to STR Fact Sheets//   0h73o g? MYHRD has 9,634 haplotypes (from 61 populations) with SWGDAM recommended loci,N 4   B=i \ i3http://www.cstl.nist.gov/biotech/strbase/y_strs.htm44H   0޽h ? ̙33___PPT10i.0nhU+D='  = @B +   0L0  p  (  ~  s *,J   x  <A6 ??    <K , URun only with minimal haplotype 0   <P/|  q7DYS19 DYS389I/II DYS390 DYS391 DYS392 DYS393 DYS385 a/b808  6T3 Ws0 6US haplotype requires 2 additional loci: DYS438 DYS439670  6H[ff '  t As of 12/5/05: 34,558 haplotypes8!0  B@  ohttp://www.yhrd.org0     0lC `  pCommercial Y-STR kits exist to amplify all of the core loci in a single reaction (plus a few additional markers)(q0V   01 p '  x*9,634 haplotypes with all US required loci2+0H  0޽h ? ̙33___PPT10i.A=+D='  = @B +  0  x(  xr x S K_ `R  j  r x S X\\   r x S <\    x <Ee -http://www.promega.com/techserv/tools/pplexy/ .0.% x <$ǍEe 0http://www.appliedbiosystems.com/yfilerdatabase/ 101(H x 0޽h ? 33___PPT10i.#+D='  = @B + 0 4\(  4x 4 c $V   4 c $4W ` <$ 0  H 4 0޽h ? ̙33___PPT10b.a+$y-D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D@' =A@BBB B0B%(D' =1:Bvisible*o3>+B#style.visibility<*4%(D' =-o6Bbox(out)*<3<*4+8+0+40 +  0 8H(  8~ 8 s *[ `   x 8 c $b    8 c $lbp 0<$ 0  ^z 0 @  8 0 @ ,$D  0f 8 63Ԕ0  @ f 8 63Ԕ0 PH 8 0޽h ? ̙33y___PPT10Y.f0+W@D'  = @B D' = @BA?%,( < +O%,( < +DA' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8$%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8$8%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*88G%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8GZ%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8Zn%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8n%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D' =%(D' =%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*8%(D4' =%(D' =%(D' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*8%(+8+0+80 +  0 <(  <x < c $l   x < <A? +??`  +x < <A@ ,??  , < < ol  c%Release "15" from 2004-12-17 16:11:24"&0%@ < 6t  Minimal Haplotype Result DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,150 f f f f f f f f < 04p >7 matches in 27,773 individuals from 236 worldwide populations2?03+ < <GF . ])Y-Chromosome Haplotype Reference Database*0*H < 0޽h ? ̙33___PPT10i.fIH+D='  = @B +  0 @M(  @~ @ s * l   4 @ <90  In cases where a Y-STR profile is observed a particular number of times (X) in a database containing N profiles, its frequency (p) can be calculated as follows: p = X/N An upper bound confidence interval can be placed on the profile s frequency using: 0X bbb$B$"ff0b B bBj @ S AA -??"?  0  - @ Bx3Ԕ0[ I7 matches in 27,7730 @ 6  b p = 7/27,773 = 0.000252 = 0.025%&!0v @ s ,AB .??"?` ` 0 . @ s *@`   v6= 0.000252 + 0.000187 = 0.000439 = 0.044% (~1 in 2270)$70#H @ 0޽h ? ̙33___PPT10i.feW+D='  = @B +  0 DT(  D~ D s *$ `*   ` D <`  In cases where the profile has not been observed in a database, the upper bound on the confidence interval is 1-a1/N where a is the confidence coefficient (0.05 for a 95% confidence interval) and N is the number of individuals in the database. p0 >b1bBbb  j  xbn D Bp y  x1-a1/N = 1-(0.05)[1/4,004] = 0.000748 = 0.075% (~1 in 1340) h=0    D B,3Ԕ`r H0 matches in 4,0040 D 6L  @If using database of 2,443, then the best you can do is 1 in 8164A0  H D 0޽h ? ̙33___PPT10i.fw&+D='  = @B +  0  H6(  H~ H s * `   x H c $tPP  H H 0޽h ? ̙33___PPT10i.fPC+D='  = @B +  0 0Lo(  L~ L s *x `   x L c $PPP  1 L <`0aZ OIssues raised by Peter de Knijff at his Promega meeting presentation (Oct 2004)8P08f f f0H L 0޽h ? ̙33___PPT10i.~g`Q +D='  = @B +  0 @PS(  P~ P s * `   x P c $ԳPp   P < q q7From his presentation at the Promega meeting (Oct 2004)808f0 P <Y ,$  0 VDatabase estimates are at most qualitative& ,0,3H P 0޽h ? ̙33___PPT10f.~g5+[D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(DD' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*P%(D' =-s6Bwipe(left)*<3<*P+8+0+P0 +n  0 }PT(  Tx T c $jz   x T c $PP   T <Ȼq q7From his presentation at the Promega meeting (Oct 2004)808fH T 0޽h ? ̙33___PPT10i.gp+D='  = @B +}  0 ` \$(  \r \ S  lO   r \ S C `   H \ 0޽h ? 33___PPT10i.a +D='  = @B +}  0  p$(  pr p S 8 `   r p S > `  H p 0޽h ? 33___PPT10i.!Z+D='  = @B +}  0  t$(  tr t S  `   r t S ° `   H t 0޽h ? 33___PPT10i.#@^+D='  = @B +  0 $G(  $~ $ s *  `   ~ $ s * `   $ < 9For more information, see: http://www.yhrd.org/index.html :0:f"H $ 0޽h ? ̙33___PPT10i.tgP+D='  = @B +6  0L0 5-0 P(  P~ P s *$ `   T P C ,A%cell-flat2gx P <8x  ? SOURCE: ? H P 0޽h ? 33___PPT10i.;+D='  = @B +  0  ](  ~  s * `   x  <A ??    <@  o9http://www.fbi.gov/hq/lab/fsc/backissu/july1999/dnaf1.htm:0:  <@   XDisadvantages of mtDNA testing: Low power of discrimination Labor intensive Expensive $Y0!80  <H[ W Advantages of mtDNA testing: Higher copy number per cell Results with highly degraded DNA Results with limited sample (hair shaft) $0gH  0޽h ? ̙33y___PPT10Y+D='  = @B +$  0 ##P YYX#(  X^ X C 6A&russianfamily-x X c $ĉ `   f )/ X C" `R X 0?p"# X C X+#  OTsarina Alexandra"  H X C oHB X C D  X C   X  OTsar Nicholas II"  HB  X C D"HB  X C DpZp*!HB  X C DH2  X C 3fo@`HB X C DZ@ZT Pp X# P"p#HB X C D<2 X # 3fPpHB X C Dp#Z%Z( X %&HB X C D$Z$zT Pp X# $z %!HB X C D<2 X # 3fPpHB X C D %*!`'*!( X `' (!HB X C D@&*!@&J"H X C 3fo%"&#HB X C D@&!@&"fB X 6D?@*!*!T    X# *!j#< X # 3f  HB  X C D@ @ T Pp !X# *! j#HB "X C D<2 #X # 3fPpHB $X C D*!J"H2 %X C 3fo J"@j#HB &X C D*!J"H2 'X C 3foPJ"pj#HB (X C D*!J"H2 )X C 3foJ"j#HB *X C Dj!j<2 +X # 3f!"HB ,X C D j ( -X `HB .X C D  fB /XB 6D?HB 0X C DHB 1X C D % :&H2 2X C o :& Z'HB 3X C D ZZ<2 4X # 0HB 5X C D Z z( 6X   HB 7X C Dp# #<2 8X #  j# $HB 9X C D # %( :X Pj#p$ N   " ;X  z j#HB X C D D ` D <2 ?X # `  HB @X C D@ D @ d!( AX   H BX C o HB CX C DHB DX C Dj@j<2 EX # @`HB FX C D j ( GX HB HX C D   IX B'IXo?' * kXenia Cheremeteff-Sfiri  JX B|1JX3fo?!%)' QPrince Philip Duke of Edinburgh    KX <4;KXoXX?B`  mGeorgij Romanov $rB LX BD? " MX 0@FMX?2%0!* `"Mitotype 16111T 16357C 263G 315.1C"#   NX 0PNX?( / 4Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C85     OX 0@\OX?pr" F16169T/C"    PX 0fPX?Zv& F16169T/C"   rB QX BD?&HB RX C DHB SX C D<2 TX #   -HB UX C D( VX  - WX BqWXo?e jLouise of Hesse-Cassel  2 XX 0<  "SOURCES: Gill et al. (1994) Nature Genetics, 6, 130 135.; Ivanov et al. (1996) Nature Genetics, 12, 417 420; Stone, R. (2004) Science, 303, 753.r# :R? YX <'@ cD.N.A. Box 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic PressTd$    ( H X 0޽h ? 33___PPT10i.4.+D='  = @B +A  0 *@"@ pph?(  h;F J)W h J) hB < S gControl region (D-loop)4 zr hB <GjJ"`-3ZB hB s *D i  hB C BCQDE FQ@= a  hB <  : :1/16,569   f hB 6 ( f  hB 6=  hB <\ 5cyt b  hB <qn 3ND5  hB <B  3ND6f  hB 6x0  f hB 6)c7f hB 6 M 0p f hB 6  * f hB 6 '  hB <uq *1  3ND4ZB h s *D  TB h c $D8\TB h c $D/ _TB h c $D>T ` f hB 6$ {G TB hB c $D  TB hB c $D9  TB hB c $D E TB hB c $DTB hB c $D o f hB 6$W  f hB 6M[  f hB 6_ G-   hB <l$   4ND4L !hB < A_ 3ND3 "hB <~j* 5COIII #hB <   4ATP6 $hB C BnCDE(F n7 -Q\e9y @    - TB %hB c $D ]  &hB <! H  4ATP8 'hB < ;   4COIIf (hB 6ɸ: c  )hB <   ; 12S rRNA   *hB <@7 ; 16S rRNA   +hB <hL 3ND1 ,hB <6 A  3ND2 -hB <j  3COIf .hB 67  f /hB 6  f 0hB 6? m f 1hB 6v = f 2hB 6] f 3hB 6  5f 4hB 6 % f 5hB 6F Oi f 6hB 6   7hB <($9 z8 : >OH  ZB 8h s *D #Z  9hB 0(  E 9-bp deletion :hB C g ;BCQDE FQ@u  ;hB <l- @  >OL  ` h s *Df |V ?h <2>   5F @h <6 Y 5VTB AhB c $DJ  TB BhB c $D G TB ChB c $D$ Dh <<;  6L1 Eh <? E  5I Fh <BHO   5QTB GhB c $D 1 TB Hh c $DZN x  Ih <G wc  5Mf JhB 64   Kh <JX 6a  5W Lh <N<i )  5A Mh <S   5N Nh <U # q  5C Oh <Y I  5Y Ph < ^  6S1 Qh <a {  5D Rh <4e^  W 5KTB Sh c $D   Th <4\  5G Uh <*   5R Vh <y 9  5H Wh <|J+ G  6S2 Xh <  6L2 Yh <:S 5E Zh < n -  5P [h <A<  5T \h s * A | 5HV1 ]h s *H  x 5HV2 ^h <Cd 516024  _h <8  516365  `h <HKH &  273  ah <~ # 3340  bh <(G 516024  ch <<3 3576  dh <  H  F 16,569 bp TB eh c $D b TB fh c $D   gh <p j  11  hh 0 ) :22 tRNAs   ih < 3 92 rRNAs jh <P :13 genes   kh 0#{ BHeavy (H) strandRB lh s *D& mh 0Х  W  BLight (L) strandRB nh s *D @ ; oh <' _Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic PressT`     (  ph 0%  E Coding RegionH h 0޽h ? 33___PPT10i.C +D='  = @B +  0 P E(  x  c $%   x  c $TJ     6 @/  _Coding Region (577-16023)* @  0N P  15,446 nucleotide positions Challenges with typing widely spaced SNPs Multiplex PCR required Polymorphisms may have medical significance<F,F,H  0޽h ? ̙33___PPT10i.LU+D='  = @B +!  0 | 70 (  z   ,$D  0z  s *C"?Ez  s *C"?zz  s *C"?Fz  s *C"? z  s *C"?l L z  s *C"?    62R~  GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT CTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTTGATAAGA 15970 15980 15990 16000 16010 16020 CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA GACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT 16030 16040 16050 16060 16070 16080 ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT TGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA 16090 16100 16110 16120 16130 16140 ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT 16150 16160 16170 16180 16190 16200 CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG 16210 16220 16230 16240 16250 16260 CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG 16270 16280 16290 16300 16310 16320 CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC GTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG 16330 16340 16350 16360 16370 16380 TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT AGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA 16390 16400 16410 16420 16430 16440  P 33 33 P 33F   5 P 33 P 33 P 33D   5 5 P 33G  5  O 33 N 33  lB   6DԔ"    C ^2  YFBI A1 (L15997).0F8  3TB  c $DoZB  s *Do  C ,e@ AHV1"CdB @ <DԔrd  <   6iz u' G C-stretch" 0  8     4  TB ( c $Do   ZB ) s *Do     * C o F  GHV1(0C + C s $@ YFBI B1 (H16391).0F - <[ fHypervariable Region I0   . < y`  A 16024-16365 0  / 01YPM@ m342 bp examined 0 R 5 0_R D Revised Cambridge Reference Sequence (rCRS)  formerly known as the  Anderson sequence(X$ * 6 <LdR 9HV1( 7 0hRVP lAdapted from Figure 10.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press@m-    ( H  0޽h ? 33E=___PPT10. ?+qD'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*+@  0  A0 (   z     ,$D  0z  s *C"?kz  s *C"?z  s *C"?z  s *C"?I  z  s *C"? Y   6$tR<E GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT TTGGTATTTT CTAGTGTCCA GATAGTGGGA TAATTGGTGA GTGCCCTCGA GAGGTACGTA AACCATAAAA 10 20 30 40 50 60 CGTCTGGGGG GTATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTC GCAGACCCCC CATACGTGCG CTATCGTAAC GCTCTGCGAC CTCGGCCTCG TGGGATACAG 70 80 90 100 110 120 GCAGTATCTG TCTTTGATTC CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATT CGTCATAGAC AGAAACTAAG GACGGAGTAG GATAATAAAT AGCGTGGATG CAAGTTATAA 130 140 150 160 170 180 ACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GCTTGTAGGA CATAATAATA TGTCCGCTTG TATGAATGAT TTCACACAAT TAATTAATTA CGAACATCCT GTATTATTAT 190 200 210 220 230 240 ACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA TGTTAACTTA CAGACGTGTC GGTGAAAGGT GTGTCTGTAG TATTGTTTTT TAAAGGTGGT 250 260 270 280 290 300 AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC ATCTCTGCCA AACCCCAAAA TTGGGGGGGA GGGGGCGAAG ACCGGTGTCG TGAATTTGTG TAGAGACGGT TTGGGGTTTT 310 320 330 340 350 360 ACAAAGAACC CTAACACCAG CCTAACCAGA TTTCAAATTT TATCTTTTGG CGGTATGCAC TGTTTCTTGG GATTGTGGTC GGATTGGTCT AAAGTTTAAA ATAGAAAACC GCCATACGTG 370 380 390 400 410 420 TTTTAACAGT CACCCCCCAA CTAACACATT ATTTTCCCCT CCCACTCCCA TACTACTAAT AAAATTGTCA GTGGGGGGTT GATTGTGTAA TAAAAGGGGA GGGTGAGGGT ATGATGATTA 430 440 450 460 470 4800 L      M B  5   M D     M C    M    N    N   N 8 E ;ETB   c $DoZB   s *DoE   C R4 AHV2"C^B  6DԔ~ d  < Wp   6R   G C-stretch" 0  8 9 P l  :P 9 l TB ! c $Do P l ZB " s *Do9 T T  # C #R `  GHV2(0C $ C (R@0 ( W FBI D1 (H408).0 FlB & 6DԔ"  ' C -R0{  W FBI C1 (L048).0 F^B 2@ 6DԔ < < 0 RYPM@ m268 bp examined 0 R ? 0\R D Revised Cambridge Reference Sequence (rCRS)  formerly known as the  Anderson sequence(X$ * @ <R 9HV2( A 0RVP lAdapted from Figure 10.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press@m-    ( H  0޽h ? ̙33E=___PPT10.`e+qD'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*+!  0 g_ "#(  z  F   ':b ,$D  0   h"Cj f b&Extract mtDNA from evidence (Q) sample '&B bB  c $Do"a   h" 3 \ PCR Amplify HV1 and HV2 Regions ! B bB  c $Do"3   Xh" w v.Sequence HV1 and HV2 Amplicons (both strands),/ B B     Hh"#P ?  m1Confirm sequence with forward and reverse strands 21B bB   c $Do"s     " F  o3Note differences from Anderson (reference) sequence 43B bB   c $Do"?  l -  # - ,$D  0   h"- 2 r6Compare with database to determine haplotype frequency 76BbB  c $D>" 2z  D0F   `j}b ,$D  0  3  " D }?Performed separately and preferably after evidence is completed"@?B     " f c'Extract mtDNA from reference (K) sample ('B bB  c $Do"CaC   ` " J3 \ PCR Amplify HV1 and HV2 Regions ! B bB  c $Do"C3C    " Jw v.Sequence HV1 and HV2 Amplicons (both strands),/ B B    x " ?  m1Confirm sequence with forward and reverse strands 21B bB  c $Do"CsC   _" 0F  o3Note differences from Anderson (reference) sequence 43B bB  c $Do"C? C ;  <|_' _Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic PressT`     ( r  S yj%    l &b   "b & ,$D  0@ ] b }  !] b }      "C E   UCompare Q and K sequences BbB  c $Do"] b E bB B c $Do"b }E   0h&   EQuestion Sample  0 ,  FReference SampleH  0޽h ? 33  ___PPT10 .;,+aD '  = @B Dm ' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*"%(D' =-o6Bwipe(up)*<3<*"D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*#%(D' =-o6Bwipe(up)*<3<*#+a 0 xpp !`(  `  ` 0d j  TCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA ACCGCTATGT ATTTCGTACA AGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT TGGCGATACA TAAAGCATGT 16030 16040 16050 16060 16070 16080 16090 16100 TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT 16110 16120 16130 16140 16150 16160 16170 16180 AAACCCCCTC CCCATGCTTA CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC TTTGGGGGAG GGGTACGAAT GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG 16190 16200 16210 16220 16230 16240 16250 16260 CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC CATTTACCGT ACATAGCACA GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG GTAAATGGCA TGTATCGTGT 16270 16280 16290 16300 16310 16320 16330 16340 TTACAGTCAA ATCCCTTCTC GTCCC AATGTCAGTT TAGGGAAGAG CAGGG 16350 16360 ATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTC GCAGTATCTG TCTTTGATTC TACGTGCG CTATCGTAAC GCTCTGCGAC CTCGGCCTCG TGGGATACAG CGTCATAGAC AGAAACTAAG 80 90 100 110 120 130 140 CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATT ACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GACGGAGTAG GATAATAAAT AGCGTGGATG CAAGTTATAA TGTCCGCTTG TATGAATGAT TTCACACAAT TAATTAATTA 150 160 170 180 190 200 210 220 GCTTGTAGGA CATAATAATA ACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCA CGAACATCCT GTATTATTAT TGTTAACTTA CAGACGTGTC GGTGAAAGGT GTGTCTGTAG TATTGTTTTT TAAAGGTGGT 230 240 250 260 270 280 290 300 AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC TTGGGGGGGA GGGGGCGAAG ACCGGTGTCG TGAATTTGTG 310 320 330 340  m 33 A  5 : 33 2 33E    33 P 33  X 33  33 x % 33 33   L        :   0 D     L  F  R /  33f ` 6Ԕ# cX  Revised Cambridge Reference Sequence (rCRS)  formerly known as the  Anderson sequence6X$ * ` < H "HV1: 16024-16365 (342 bp examined)6#0  ` <dR  HV2: 73-340 (268 bp examined)60    ` <Cm 9HV1 !` <0   9HV2H ` 0޽h ? 33___PPT10i. ?+D='  = @B +  0 d\ (    <Rj* (^  6Ul  Pb5 P b5,$D  0.  H RS BACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT CC2 ~} | 5  BRPb  16090 16100 16110 16120 16130 16140     B@R  8rCRSj   H Sm BACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT RC &2 ~} |    B(j 5Q   Ba5 5Kj   HmS- BACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT RC &2 ~} | ZB  s *Db}"}  00  nmtDNA sequences from tested samples are aligned with the reference rCRS sequence (e.g., positions 16071-16140)(oPl  "  ",$D  0  0v. (  PSample Q 16093C 16129A   0h  PSample K 16093C 16129A   0(h "V  YDifferences are reported by the position and the nucleotide change (compared to the rCRS) ZBH  <h' lAdapted from Figure 10.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic PressTm-    ( x  c $h `  h fl \  \ ,$D  0q@ \`  \` l  <oB  <h\`  916093q@    l  <ok  <h  916129H  0޽h ? 33  ___PPT10 .0d+mD '  = @B D ' = @BA?%,( < +O%,( < +D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwipe(up)*<3<*D' =%(D' =%(D7' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-s6Bwipe(left)*<3<*+}  0  l$(  lr l S D2 `   r l S  `   H l 0޽h ? 33___PPT10i.+V+D='  = @B +q  0  (  F $  #   <nA ??"`     s *(N   = 16093 (C/T)    B   16086  B  $  16101V  C A"`  `B  0DԔZ T Z  BF PjP    P   <yA ??"`Pjr V   C ՄA"`PP^   6 @ `'   <' _Figure 10.9, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press@`     ( r  S | `   H  0޽h ? 33___PPT10i.q)+D='  = @B +   0 `  (  ~  s *l `   x  c $D[{  z P    P  ,$D  0J  # +A"`P : 6  <P   Review forward and reverse sequences across 610 bases only to report& F0F@z  :{   :{,$D 0  <Ŋ  > 263G, 315.1C    0@ʊP :{ ZMost common type: found in ~7% of Caucasians& .0.fH  0޽h ? ̙334,___PPT10 .oa+qD'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D7' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-s6Bwipe(left)*<3<*D' =%(D' =%(D3' =4@BBBB%(D' =1:Bvisible*o3>+B#style.visibility<*%(D' =-o6Bwheel(8)*<3<*+  0 p <(  x  c $LԊ   x  c $$ՊJj     6يL kR  aScreening assays are essentially a presumptive test prior to final confirmatory DNA sequencing. b0bf  0ފ  eSequencing is necessary to certify that every position matches between a question and a known sample.f0fH  0޽h ? ̙33___PPT10i.kA+D='  = @B +D  0 ?? wwO?(    0b6 (  <T R  @Reported Types  < V ' TK: 1-1-1-1-1-1-1-1-1-1   <WA w iQ: 1-2-3-2-0-1-4-2-2-w14q8F     w `  0+T   #    <wg 2IA   <wHP 11   <(wP 124 N `l   `lZB   s *DԔ`ZB  s *DԔ6`6ZB  s *DԔ`ZB  s *DԔB BZB  s *DԔ ZB  s *DԔO OZB  s *DԔ ZB  s *DԔ\`\ZB  s *DԔ`ZB  s *DԔ) )ZB  s *DԔ ZB  s *DԔi i ZB  s *DԔ  ZB  s *DԔv v ZB  s *DԔ ` ZB  s *DԔ ` ZB  s *DԔ  ZB  s *DԔ  ZB  s *DԔ  ZB   s *DԔ  ZB ! s *DԔ" "ZB " s *DԔ ZB # s *DԔ`ZB $ s *DԔF`FZB % s *DԔ`ZB & s *DԔR`RZB ' s *DԔ ZB ( s *DԔ_ _ZB ) s *DԔ`ZB * s *DԔl`lZB + s *D)  , 0w| 2IC - <hw g 2ID . <Dwh d  2IE / 0w  g 3IIA 0 <\w $ 3IIB 1 <$g 3IIC 2 <hx 3IID 3 <H 3189 4 < 516093 5 0(0  5HVI 6 <  6HVIIrb 7 BGo  rb 8 BGo P 9 <L^` 11 : <h` 12 ; <`  11 < <n`   12 = <`  13 > <`2  14 ? <h` 11 @ <~0 ` 12 A < `  11 B < `B  12 C <hs @ ` 11 D <p&  ` 12 E < `R  11 F <X0 `  12 G <z `r  13 H <. `  14 I <@ `p  15 J < œ@`  16 K <Ǔn ` 11 L <̓` 12 M <(Г2` 14 N <ӓ` 15 O <Pד~`0  11 P <ړ`  12 Q <xޓB` 11 R <  ` 12 S < `  13 T <4p"` 13 U <`2  3RefZB V s *DԔ0 0 W <\`  17ZB X s *D)ZB Y s *D)ZB Z s *D)ZB [ s *D)00ZB \ s *D)ppZB ] s *D)` ` ZB ^ s *D)  ZB _ s *D)  ZB ` s *D)ZB a s *D)ZB b s *D) c <h 5K` d 0! ZB e s *D)( ZB f s *D)( ZB g s *D)@(@ ZB h s *D)P(P ZB i s *D)( ZB j s *D) (  ZB k s *D) (  ZB l s *D)P(P ZB m s *D)`(` lB n <DA)(  o <Z1p  5Q p BZ  (ZB q s *D~  f r 6Z9 l  s 0Z = r  @ blank ( t < Z' `Figure 10.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press@a!    ( r u S 88   8  v <h! !  ; Weak signal \ w 6UԔF C ,$D 0 pIf known (K) and question (Q) samples do not match, there is no need to involve the expense of mtDNA sequencing qq_ H  0޽h ? 33___PPT10b.0d+D'  = @B D' = @BA?%,( < +O%,( < +D' =%(D' =%(D@' =A@BBBB0B%(D' =1:Bvisible*o3>+B#style.visibility<*w%(D' =-o6Bwheel(4)*<3<*w+8+0+w0 +  0  0v(  0~ 0 s *Z)  Z x 0 <A ??y`   0 0Z`Z /Melton et al. (2005) J. Forensic Sci. 50: 73-80J00ffff Ff9 0 0tZz Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669.*0 0 s * Z Z0 VMelton et al. (2005) obtained a full or partial mtDNA profile for >92% of hairs tested.W0JH 0 0޽h ? ̙33y___PPT10Y+D=' = @B +   0   4N (  4x 4 c $*Z``PP  Z x 4 <A ??P0@  x 4 <A! ?? x 4 <A" ??Yp P  4 <H-Z3Ԕ I108 bp size reduction0 4 <h1Z3Ԕ|` I160 bp size reduction0^ 4 6fԔ P ^  4 6Ԕpp0 ^  4 6fԔ @ ^  4 6Ԕ @  4 B6Z p  <Hellmann, et al. (2001) Int. J. Legal Med. 114(4-5): 269-273F=0      X2  4 03X2 4 03 @ LB 4@ c $D3@ X2 4 03p X2 4 03  LB 4@ c $D3ppp  4 0(@Zf> j* d,First use of miniSTRs for typing hair shafts-0-H 4 0޽h ? ̙33y___PPT10Y+D=' = @B +Z 0 p\j(  \d \ c $XWK   l \ s *6lX %  l T@Figure 9.2 Schematic illustrating the types of autosomal or Y-STR profiles that might be observed with sexual assault evidence where mixtures of high amounts of female DNA may mask the STR profile of the perpetrator. Y-STR testing permits isolation of the male component without having to perform a differential lysis. H \ 0`jڍ ? 3380___PPT10. %0   0 @(  d  c $XWK   l  s *?lX %  l *Figure 9.5 Schematic illustration of how multiple PCR primer binding sites give rise to multi-copy PCR products for (A) DYS385 a/b and (B) DYS389I/II. Arrows represent either forward  F or reverse  R primers. In the case of DYS385 a/b, the entire region around the STR repeat is duplicated and spaced about 40,775 bp apart on the long arm of the Y-chromosome. Thus, amplification with a single set of primers gives rise to one peak if the  a repeat region is equal in size to the  b repeat region or separate peaks if  a and  b differ in length. DYS389 possess two primary repeat regions that are flanked on one side by a similar sequence. Widely used forward primers bind adjacent to both repeats generating amplicons that differ in size by ~120 bp. Note that DYS389II is inclusive of the DYS389I repeat region and therefore some analyses subtract DYS389II-DYS389I repeats.,<H  0`jڍ ? 3380___PPT10.; 0 91<(  <d < c $XWK   l% < s *dFlX %  l Figure 9.1 Illustration of inheritance patterns from recombining autosomal genetic markers and the lineage markers from the Y-chromosome and mitochondrial DNA.H < 0`jڍ ? 3380___PPT10.~ 0 xp D(  Dd D c $XWK   ld D s *TZlX %  l Figure 9.3 An example pedigree showing patrilineal inheritance where all shaded males have the same Y-chromosome barring any mutations. To help identify the person in question, any of the other males with the same patrilineage could provide a reference sample to assist in a missing persons investigation, mass disaster victim identification, or deficient paternity test (boxed region) where the father is deceased or not available for testing.' H D 0`jڍ ? 3380___PPT10.w@\ 0 0 Ll(  Ld L c $XVJ   l L s *0hlX %  l VBFigure 2.3 The human genome contained in every cell consists of 23 pairs of chromosomes and a small circular genome known as mitochondrial DNA. Chromosomes 1-22 are numbered according to their relative size and occur in single copy pairs within a cell s nucleus with one copy being inherited from one s mother and the other copy coming from one s father. Sex-chromosomes are either X,Y for males or X,X for females. Mitochondrial DNA is inherited only from one s mother and is located in the mitochondria with hundreds of copies per cell. Together the nuclear DNA material amounts to over 3 billion base pairs (bp) while mitochondrial DNA is only about 16,569 bp in length.H L 0`jڍ ? 3380___PPT10.O@ 0  d(  dd d c $XVJ   l d s *vlX %  l TFigure 9.10 Ancestry of Thomas Jefferson and Eston Hemings male lines. The shaded boxes represent the samples tested by Foster et al. (1998) in their Jefferson Y chromosome study. A male descendant of Eston Hemings, son of Thomas Jefferson s slave Sally Hemings, was found to have a Y chromosome haplotype that matched male descendants of Field Jefferson, President Thomas Jefferson s uncle. A male descendant of Thomas Woodson, claimed by some to be descended from Jefferson, had a different Y haplotype and therefore could not have been a Jefferson.$+H d 0`jڍ ? 3380___PPT10.MG 0  l(  ld l c $XWJ   l l s *lX %  l |hFigure 10.1 Schematic showing the circular mitochondrial DNA genome (mtGenome). The heavy (H) strand is represented by the outside line and contains a higher number of C-G residues than the light (L) strand. The 37 RNA and protein coding gene regions are abbreviated around the mtGenome next to the strand from which they are synthesized (see Table 10.2). Most forensic mtDNA analyses presently examine only HV1 and HV2 in the non-coding control region or displacement loop shown at the top of the figure. Due to insertions and deletions that exist around the mtGenome in different individuals, it is not 16,569 bp.H l 0`jڍ ? 3380___PPT10.F 0   (  d  c $XWJ   l  s *lX %  l TFigure 10.4 Process for evaluation of mtDNA samples. The evidence or question (Q) sample may come from a crime scene or a mass disaster. The reference or known (K) sample may be a maternal relative or the suspect in a criminal investigation. In a criminal investigation, the victim may also be tested and compared to the Q and K results. T UH  0`jڍ ? 3380___PPT10.b 0 jb (  d  c $XWJ   lV  s *LlX %  l Figure 10.9 (A) Sequence heteroplasmy at position 16093 possessing both C and T nucleotides compared to (B) the same region (positions 16086-16101) on a different sample containing only a T at position 16093.H  0`jڍ ? 3380___PPT10.@ 4 0  "(  d  c $XWJ   l~  s *lX %  l Figure 10.10 (A) Results schematically displayed of a known (K) reference and a question (Q) sample that do not match one another using the Roche LINEAR ARRAY mtDNA HVI/HVII Region-Sequence Typing Strips. (B) Types are reported as a string of numbers representing the LINEAR ARRAY probe results. Failure of the PCR product to bind to a probe region (e.g., HVIE in sample Q) is referred to as a  blank , is reported as a zero in the string of numbers, and is due to polymorphisms in the sample near the probe site that disrupt hybridization. Weak signals such as indicated by the arrow for 189 in sample Q are also due to a closely spaced polymorphism that disrupts full hybridization of the PCR product to the sequence-specific probe present on the LINEAR ARRAY. H  0`jڍ ? 3380___PPT10.D 0  5(  d  c $XVJ   l  s *8lX %  l  Figure 10.6H  0`jڍ ? 33 0  5(  d  c $XVJ   l  s *lX %  l  Figure 10.6H  0`jڍ ? 33 0 yq  (  d  c $XWJ   he  s *lX %  h Figure 10.8 (A) Comparison of sequence alignments for hypothetical Q and K samples with (B) conversion to the revised Cambridge Reference Sequence (rCRS) differences for reporting purposes.&H  0`jڍ ? 3380___PPT10. 0  d5(  dd d c $XVJ    d s *X %    Figure 10.6H d 0`jڍ ? 33xx Oվ&5%eJ$5$ce!TRJ(N+ %E2D2ԉgixw>kwX{ ̽}B;"QR+rA$9 $Dwyk.YRpPJR>@$7|O ?( +@!Pb8(WA)PeA9p5( ZP T5u?PoۑHmPz&P47 -4Mm)4A 6-hڃ;@p':Π =A/p/ > 0 gϻO.a|ЖK#Mފ$x!)$jg&F$T _翗:O,=SjoߩKAr8?^;NEPI?A]'Ze3tT3B+t`spz&m8m?ӖӼ .Uۏqi' w>ްϿh~hn0&֍Ru_]:w!Mnl0=o炿Mq\+\ q+wi㺾15Pϣ*e;%E>d[KէӘ4 [ M~?~1iP U_ؙ\[m5[B"rmS,C3̒ӽf(qW0OQ`4x<ƀoi x< E&` ^ST0 Uxf7[`6?0oy.!X,'SXe`9+K kZ|փ `#|x- 8#8 ~G+8 dRA \.% +. @n@~PWB0( @qP\ %R4(ʂrjP\*A%PTUj &j:FP78n4n1hnMhZh mA;:;AG t]@W  z{Aog~?@/IaQ#HF'S`  πgsy0^/`&e0L ^`xfs\6`x>#1XO3,r9XV/`X ր` F | 6-`+l- 8#8 ~G+8 dRA$\.% +. @n@~PWB0( @qP\ %R4(ʂrjP\*A%PTUj &j:FP7hn1hnMhZh mA;:;AG t]@W  z{Aog~?@/` Gc`8x#` O1`,x<ρ8xƃ `"&0L3+`&x^oYM s`x =>|`X >,`X _U`uW`=6Mkl[7`v` o^Ap|?a8~_Qp $ Ҁ$w޳ "p1d2   PE(J+U$wP e@YP\ ʃk@"***TA pOp=@mPz&P47 -4Mm)4A 6-hڃ;@p':Π =A/p/ > 0 C0(x `$x)0Og9<^/x0Ld2it`x 0 9`.xw|B,'SXe`9+K kZ|փ `#|` v`x? 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" # $ % & ' ( ) * + , - . / 0 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` a b c d e f g h i j k l m n o p q r s t u v w x y z { | } ~        !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~=Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImagePowerPoint Document(`DocumentSummaryInformation8 ,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image+%Bitmap Image Paint.Picture0Bitmap Image,&Bitmap Image Paint.Picture0Bitmap Image-'Equation Equation.30,Microsoft Equation 3.0.(Equation Equation.30,Microsoft Equation 3.0Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageNhttp://www.familytreedna.com/index.htmlNhttp://www.familytreedna.com/index.htmlDhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jsphttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image/ 0DArial NewripthhT=v  0"DTimes New RomanhhT=v  0 DBazookaw RomanhhT=v  00DSymbolw RomanhhT=v  0@DFreestyle ScripthT=v  0BPDCourier NewripthT=v  01`DImpact NewripthT=v  0"@ .  @n?" dd@  @@`` J    $ S4RG#')F*>+"/&34I-89 $ &MNO P QRSTU ;;;: " @  #y= +ROqVO$'3:,x\ 8B, [ Z" /X R$ͪ2(S"/梢U b$`- 9+3񓶑+VP b$ೣ K(Q_#kR$lUU |<(y8b$Hn>=T# )ab$V yPqa@nb$Ô1OXJ$$R$,JL=H~Z!b$w Fg˄]f1R$V`2| # b$J+ Yy!Sc5b$v03'"esR$Bk~ꍣfq$b$|bp,VA;b$   !b$Àpw&8s r$$E2fY_,I!)i b$2L7Sށ7{ b$,epzKu2 $$b$ljVQ|`{ b$=Zx ]X~rZ^ $b$'&.q b$M.` ~~DX0 b$;}k57T $$b$FD: 2QUn b$aMg$lC%j b$iewu`ZW $$b$LQ }C9F,w4; R$: ]=p $b$ 6Sr:YV2= $$$$b$Z%hu'z b$]0sgۄ,iO s b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g40d0dv  0ppp@ <4dddd4w 0h0=v g4ddddtv  02pn p <4BdBd4x 0h<80___PPT10 ?  %H#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs,-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5& Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR Kits PowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)x ddd  o   Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)\dd  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> # Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate, K *Duplications and Deletions of Y-Chromosome++Y-STR Mixtures8Statistical treatment is not the same as autosomal STRs ,) ~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples(( #Differences from Reference SequenceChallenges with mtDNAsData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes DNA Database Quality66*6* T'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88  &2A Common Use of mtDNA Analysis is for Hair ShaftsRPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSf/   rLp1xu(<  D mv,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image+%Bitmap Image Paint.Picture0Bitmap Image,&Bitmap Image Paint.Picture0Bitmap Image-'Equation Equation՜.+,D՜.+,     S On-screen ShowNISTree9{ CArialTimes New RomanBazookaSymbolFreestyle Script Courier NewImpactDefault Design Bitmap ImageMicrosoft Equation 3.0$Advanced Topics in STR DNA AnalysisOutline for This Section.Human Genome 23 Pairs of Chromosomes + mtDNA5Role of Y-STRs and mtDNA Compared to Autosomal STRsDifferent Inheritance Patterns"Lineage Markers: Y-STRs and mtDNAOY-STRs permit extension of possible reference samples in missing persons casesSlide 8Value of Y-Chromosome MarkersTHE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612 Slide 11(What has happened in the past few years"Disadvantages of the Y-ChromosomeForensic Advantages of Y-STRs2Scenarios Where Y-STRs Can Aid Forensic Casework Slide 16%PowerPlex Y Performance in Our HandsSelection of Core Y-STR LociCore Y-STR Characteristics Slide 20 Slide 21 Slide 22 Slide 23 Slide 242New Y-Chromosome Information Resources on STRBase1Y-Chromosome Haplotype Reference Database (YHRD)#Haplotype Databases for Y-STR Kits!Statistics with Y-STR HaplotypesExample Y-STR Haplotype www.YHRD.org:Frequency Estimate Calculations Using the Counting Method2When there is no match with the counting method$The Meaning of a Y-Chromosome MatchDifficult Questions!Conclusions from Peter de Knijff0What Peter de Knijff Reports with a Y-STR Match>Can Y-STR results be combined with autosomal STR information?(International Forensic Y-User WorkshopsMitochondrial DNA (mtDNA),Comparison of Nuclear and Mitochondrial DNA8Identifying the Romanov Remains (the Last Russian Czar) Slide 42Control Region (16024-576) Slide 44 Slide 45(Process for Evaluation of mtDNA Samples Slide 47$Differences from Reference SequenceChallenges with mtDNA(Sequence Heteroplasmy at Position 16093Disadvantages to Sequencing Advantages to Screening Methods8LINEAR ARRAY mtDNA Typing Strips: New Screening Method1A Common Use of mtDNA is for Hair Shaft AnalysisSPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsmtDNA and miniSTRsTHANK YOU FOR YOUR ATTENTION  Fonts UsedDesign TemplateEmbedded OLE Servers Slide Titles9 8@ _PID_HLINKSAhttp://www.seattle.com/(http:/.30,Microsoft Equation 3.0.(Equation Equation.30,Microsoft Equation 3.0Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageNhttp://www.familytreedna.com/index.htmlNhttp://www.familytreedna.com/index.htmlDhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jsphttp://www.relativegenetics.com/servlet/moonshine/goto?page_urs b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g40d0dv  0ppp@ <4dddd4w 0h0=v g4ddddtv  02pn p <4BdBd4x 0h<80___PPT10 ?  %RL#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs,-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5& Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR Kits PowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)x ddd  o   Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)\dd  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> # Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate, K *Duplications and Deletions of Y-Chromosome++Y-STR Mixtures8Statistical treatment is not the same as autosomal STRs ,) ~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples(( #Differences from Reference SequenceChallenges with mtDNAsData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes DNA Database Quality66*6* T'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88  &0A Common Use of mtDNA is for Hair Shaft Analysis 1  #Human hair shafts contain very little DNA but because mtDNA is in higher copy number it can often be recovered Melanin found in hair is a PCR inhibitor Important Publications: Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669. Tissue grinding method described by FBI Lab Melton et al. (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. J. Forensic Sci. 50(1): 73-80. Obtained a full or partial mtDNA profile for >92% of hairs testedZZZ,ZpZBZ,bB  P6 +"RPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSf/   y  0  00(  0~ 0 s *Zu$  Z r 0 S ({ `k  H 0 0޽h ? ̙33y___PPT10Y+D=' v = @B +rp\p1u(<  D mv,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image+%Bitmap Image Paint.Picture0Bitmap Image,&Bitmap Image Paint.Picture0Bitmap Image-'Equation Equation.30,Microsoft Equation 3.0.(Equation Equation.30,Microsoft Equation 3.0Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageNhttp://www.familytreedna.com/index.htmlNhttp://www.familytreedna.com/index.htmlDhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jsphttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image/ 0DArial NewripthhT=v  0"DTimes New RomanhhT=v  0 DBazookaw RomanhhT=v  00DSymbolw RomanhhT=v  0@DFreestyle ScripthT=v  0BPDCourier NewripthT=v  01`DImpact NewripthT=v  0"@ .  @n?" dd@  @@`` J    $ S4RG#')F*>+"/&34I-89 $ &MNO P QRSTU ;;;: " @  #y= +ROqVO$'3:,x\ 8B, [ Z" /X R$ͪ2(S"/梢U b$`- 9+3񓶑+VP b$ೣ K(Q_#kR$lUU |<(y8b$Hn>=T# )ab$V yPqa@nb$Ô1OXJ$$R$,JL=H~Z!b$w Fg˄]f1R$V`2| # b$J+ Yy!Sc5b$v03'"esR$Bk~ꍣfq$b$|bp,VA;b$   !b$Àpw&8s r$$E2fY_,I!)i b$2L7Sށ7{ $$$b$ljVQ|`{ b$=Zx ]X~rZ^ $b$'&.q b$M.` ~~DX0 b$;}k57T $$b$FD: 2QUn b$aMg$lC%j b$iewu`ZW $$b$LQ }C9F,w4; R$: ]=p $b$ 6Sr:YV2= $$$$b$Z%hu'z b$]0sgۄ,iO s b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g40d0dv  0ppp@ <4dddd4w 0h0=v g4ddddtv  02pn p <4BdBd4x 0h<80___PPT10 ?  %tL#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs,-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5& Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR Kits PowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)x ddd  o   Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)\dd  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> # Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate, K *Duplications and Deletions of Y-Chromosome++Y-STR Mixtures8Statistical treatment is not the same as autosomal STRs ,) ~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples(( #Differences from Reference SequenceChallenges with mtDNAsData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes DNA Database Quality66*6* T'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88  &0A Common Use of mtDNA is for Hair Shaft Analysis 1  =Human hair shafts contain very little DNA but because mtDNA is in higher copy number it can often be recovered and successfully analyzed Melanin found in hair is a PCR inhibitor Important Publications: Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669. Tissue grinding method described by FBI Lab Melton et al. (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. J. Forensic Sci. 50(1): 73-80. Obtained a full or partial mtDNA profile for >92% of hairs testedZZZ,ZpZBZ,bB  P6 +"RPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSf/   y  0  00(  0~ 0 s *Zu$  Z r 0 S ({ `k  H 0 0޽h ? ̙33y___PPT10Y+D=' v = @B +r_^`1v(<  D mv,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image+%Bitmap Image Paint.Picture0Bitmap Image,&Bitmap Image Paint.Picture0Bitmap Image-'Equation Equation.30,Microsoft Equation 3.0.(Equation Equation.30,Microsoft Equation 3.0Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageNhttp://www.familytreedna.com/index.htmlNhttp://www.familytreedna.com/index.htmlDhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jsphttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image/ 0DArial NewripthhT=v  0"DTimes New RomanhhT=v  0 DBazookaw RomanhhT=v  00DSymbolw RomanhhT=v  0@DFreestyle ScripthT=v  0BPDCourier NewripthT=v  01`DImpact NewripthT=v  0"@ .  @n?" dd@  @@``  K    $ S4RG#')F*>+"/&34I-89 $ &MNO P QRSTU ;;;: " @  #y= +ROqVO$'3:,x\ 8B, [ Z" /X R$ͪ2(S"/梢U b$`- 9+3񓶑+VP b$ೣ K(Q_#kR$lUU |<(y8b$Hn>=T# )ab$V yPqa@nb$Ô1OXJ$$R$,JL=H~Z!b$w Fg˄]f1R$V`2| # b$J+ Yy!Sc5b$v03'"esR$Bk~ꍣfq$b$|bp,VA;b$   !b$Àpw&8s r$$E2fY_,I!)i b$2L7Sށ7{ $$$b$ljVQ|`{ b$=Zx ]X~rZ^ $b$'&.q b$M.` ~~DX0 b$;}k57T $$b$FD: 2QUn b$aMg$lC%j b$iewu`ZW $$b$LQ }C9F,w4; R$: ]=p $b$ 6Sr:YV2= $$$$b$Z%hu'z b$]0sgۄ,iO s b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g40d0dv  0ppp@ <4dddd4w 0h0=v g4ddddtv  02pn pl=/index.jspBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image <4BdBd4x 0h<80___PPT10 ?  %M#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs,-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5& Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR Kits PowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)x ddd  o   Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)\dd  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> # Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate, K *Duplications and Deletions of Y-Chromosome++Y-STR Mixtures8Statistical treatment is not the same as autosomal STRs ,) ~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples(( #Differences from Reference SequenceChallenges with mtDNAData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes Similar issues to Y-STRs but takes longer to generate mtDNA data than Y-STR haplotypes DNA Database Quality`6W6WP R 'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88  &0A Common Use of mtDNA is for Hair Shaft Analysis 1  =Human hair shafts contain very little DNA but because mtDNA is in higher copy number it can often be recovered and successfully analyzed Melanin found in hair is a PCR inhibitor Important Publications: Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669. Tissue grinding method described by FBI Lab Melton et al. (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. J. Forensic Sci. 50(1): 73-80. Obtained a full or partial mtDNA profile for >92% of hairs testedZZZ,ZpZBZ,bB  P6 +"RPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSfAcknowledgments and Questions/   }  0  l$(  lr l S D2 `   r l S  `   H l 0޽h ? 33___PPT10i.+V+D=' v = @B +$  0 $(  r  S 蜦 `   r  S + `  H  0޽h ? 3380___PPT10.?PXra[`1~v(<  D v,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Root EntrydO)@3mx PicturesCurrent User5>SummaryInformation(T      !"#$%&:<./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~-    ;w>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuv ~yz{|},  !"#$%&'()*+,-./01234/www.familytreedna.com/index.htmlKhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspurl=/index.jsp"_ "JohnButlerJohnButlerButlerBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image/ 0DArial NellD"ԥ0:A 0"DTimes New Romanԥ0:A 0 DBazookaw Romanԥ0:A 00DSymbolw Romanԥ0:A 0@DFreestyle Scriptԥ0:A 0BPDCourier Newriptԥ0:A 01`DImpact Newriptԥ0:A 0"@ .  @n?" dd@  @@`` (J    $ S4RG#')F*>+"/&34I-89 $ &MNO P QRSTU ;;;: " @  #y= +ROqVO$'3:,x\ 8B, [ Z" /X R$ͪ2(S"/梢U b$`- 9+3񓶑+VP b$ೣ K(Q_#kR$lUU |<(y8b$Hn>=T# )ab$V yPqa@nb$Ô1OXJ$$R$,JL=H~Z!b$w Fg˄]f1R$V`2| # b$J+ Yy!Sc5b$v03'"esR$Bk~ꍣfq$b$|bp,VA;b$   !b$Àpw&8s r$$E2fY_,I!)i b$2L7Sށ7{ $$$b$ljVQ|`{ b$=Zx ]X~rZ^ $b$'&.q b$M.` ~~DX0 b$;}k57T $$b$FD: 2QUn b$aMg$lC%j b$iewu`ZW $$b$LQ }C9F,w4; R$: ]=p $b$ 6Sr:YV2= $$$$b$Z%hu'z b$]0sgۄ,iO s b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g4HdHd:A 0ppp@ <4dddd0$ 0l"g4dddd:A 02pn p <4BdBd0 # 0l<80___PPT10 ?  %HM#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR KitsPowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)p   o  Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)V  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples((#Differences from Reference SequenceChallenges with mtDNAData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes Similar issues to Y-STRs but takes longer to generate mtDNA data than Y-STR haplotypes DNA Database Quality`6W6W'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88 0A Common Use of mtDNA is for Hair Shaft Analysis 1  =Human hair shafts contain very little DNA but because mtDNA is in higher copy number it can often be recovered and successfully analyzed Melanin found in hair is a PCR inhibitor Important Publications: Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669. Tissue grinding method described by FBI Lab Melton et al. (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. J. Forensic Sci. 50(1): 73-80. Obtained a full or partial mtDNA profile for >92% of hairs testedZZZ,ZpZBZ,bB  ,B RRPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSfmtDNA and miniSTRsDue to the higher copy number, mtDNA will still have a role in many highly degraded DNA scenarios or where limited biological material is present, such as hair shafts. However, miniSTRs will most likely extend the range of cases where highly informative STR data can be obtained :THANK YOU FOR YOUR ATTENTION&  Thank you for attending and participating in this Advanced Topics in STR DNA Analysis Workshop Feel free to contact us if you have further questions: John Butler (NIST): john.butler@nist.gov Bruce McCord (FIU): mccordb@fiu.edu2ZNZN*B 0/    -K0 z0 P(  P~ P s *l `   T P C ,A%cell-flat2gxH P 0޽h ? 33___PPT10i.;+D=' = @B +$   $(  r  S o `   r  S ܞ `  H  0޽h ? 3380___PPT10.mP $  $(  r  S   `   r  S   `   H  0޽h ? 3380___PPT10.?PXrQS XUm4Zv((  D v,http://www.seattle.com.http://www.seattle.com/Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image+%Bitmap Image Paint.Picture0Bitmap Image,&Bitmap Image Paint.Picture0Bitmap Image-'Equation Equation.30,Microsoft Equation 3.0.(Equation Equation.30,Microsoft Equation 3.0Bitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageNhttp://www.familytreedna.com/index.htmlNhttp://www.familytreedna.com/index.htmlDhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jsphttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap ImageBitmap Image Paint.Picture0Bitmap Image/ 0DArial NellD"ԥ0:A 0"DTimes New Romanԥ0:A 0 DBazookaw Romanԥ0:A 00DSymbolw Romanԥ0:A 0@DFreestyle Scriptԥ0:A 0BPDCourier Newriptԥ0:A 01`DImpact Newriptԥ0:A 0"@ .  @n?" dd@  @@`` 0K    $ S4RG#')F*>+"/&34I-89 $ &MNO P QRSTU ;;;: " @  #y= +ROqVO$'3:,x\ 8B, [ Z" /X R$ͪ2(S"/梢U b$`- 9+3񓶑+VP b$ೣ K(Q_#kR$lUU |<(y8b$Hn>=T# )ab$V yPqa@nb$Ô1OXJ$$R$,JL=H~Z!b$w Fg˄]f1R$V`2| # b$J+ Yy!Sc5b$v03'"esR$Bk~ꍣfq$b$|bp,VA;b$   !b$Àpw&8s r$$E2fY_,I!)i b$2L7Sށ7{ $$$b$ljVQ|`{ b$=Zx ]X~rZ^ $b$'&.q b$M.` ~~DX0 b$;}k57T $$b$FD: 2QUn b$aMg$lC%j b$iewu`ZW $$b$LQ }C9F,w4; R$: ]=p $b$ 6Sr:YV2= $$$$b$Z%hu'z b$]0sgۄ,iO s b$vxP=ftVg(!G) b$m5+X [J $$$$b$qn=P"S";USed b$LQ'!TyhPL$$$$$$$$b$V0,T_`/{yr NTb$Db 7yܢ2$yed)y2$2KΌUH| 0AA`PP@ʚ;ʚ;g4HdHd:A 0ppp@ <4dddd0$ 0l"g4dddd:A 02p p <4BdBd0 # 0l<80___PPT10 ?  %HM#Advanced Topics in STR DNA Analysis$$\AAFS 2006 Workshop #6 Seattle, WA February 20, 2006 Dr. John M. Butler Dr. Bruce R. McCordB]P53fOutline for This Section Role of Y-STRs and mtDNA compared to autosomal STRs Advantages and disadvantages of lineage markers Y-STR core loci and available kits Y-STR haplotype databases and statistics mtDNA characteristics Efforts to resolve common types Hair shaft analysis with mtDNA and STRs-Human Genome 23 Pairs of Chromosomes + mtDNA. 4Role of Y-STRs and mtDNA Compared to Autosomal STRs5Autosomal STRs provide a higher power of discrimination and are the preferred method whenever possible Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings) Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quantity samples such as hair shaftsZ<3sDifferent Inheritance Patterns!Lineage Markers: Y-STRs and mtDNA""  Advantages Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy) Family members have indistinguishable haplotypes unless mutations have occurred *   Disadvantages Lower power of discrimination due to no genetic shuffling with recombination Family members have indistinguishable haplotypes unless mutations have occurred*NY-STRs permit extension of possible reference samples in missing persons casesOOO Value of Y-Chromosome Markers M THE HUMAN Y CHROMOSOME: AN EVOLUTIONARY MARKER COMES OF AGE MarkA.Jobling & ChrisTyler-Smith Nature Reviews Genetics (2003) 4, 598-612t=$fff fECUntil recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes  rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up. 0N L'What has happened in the past few years(( p Full Y-chromosome sequence became available in June 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 databases generated with thousands of Y-STR haplotypes Forensic casework demonstration of value of Y-STR testing along with court acceptanceP<[Y<P !Disadvantages of the Y-Chromosome"" Loci are not independent of one another and therefore rare random match probabilities cannot be generated with the product rule; must use haplotypes (combination of alleles observed at all tested loci) Paternal lineages possess the same Y-STR haplotype (barring mutation) and thus fathers, sons, brothers, uncles, and paternal cousins cannot be distinguished from one another Not as informative as autosomal STR results More like addition (10 + 10 + 10 = 30) than multiplication (10 x 10 x 10 = 1,000)<Rz,RQ Forensic Advantages of Y-STRsMale-specific amplification extends range of cases accessible to obtaining probative DNA results (e.g., fingernail scrapings, sexual assault without sperm) Technical simplicity due to single allele profile; can potentially recover results with lower levels of male perpetrator DNA because there is not a concern about heterozygote allele loss via stochastic PCR amplification; number of male contributors can be determined Courts have already widely accepted STR typing, instrumentation, and software for analysis (Y-STR markers just have different PCR primers) Acceptance of statistical reports using the counting method due to previous experience with mtDNAj1.^;'R1Scenarios Where Y-STRs Can Aid Forensic Casework2 "Sexual assaults by vasectomized or azoospermic males (no sperm left behind for differential extraction) Extending length of time after assault for recovery of perpetrator s DNA profile (greater than 48 hours) Fingernail scrapings from sexual assault victims Male-male mixtures Other bodily fluid mixtures (blood-blood, skin-saliva) Gang rape situation to include or exclude potential contributorsH        cST$PowerPlex Y Performance in Our Hands%%\Selection of Core Y-STR Loci ^Core Y-STR Characteristics_ g( h)m.1New Y-Chromosome Information Resources on STRBase22 0Y-Chromosome Haplotype Reference Database (YHRD)11 "Haplotype Databases for Y-STR KitsPowerPlex Y 1311 Caucasians 325 Asians 894 Hispanics 1108 African Americans 366 Native Americans -------------- 4,004 total (as of March 2005)p   o  Yfiler 1276 Caucasians 330 Asians 597 Hispanics 985 African Americans 106 Native Americans 105 Filipino 59 Sub-Saharan Africans 103 Vietnamese --------------- 3,561 total (as of December 2004)V  yB Statistics with Y-STR Haplotypes!!6Most labs will probably go with the counting method (number of times a haplotype is observed in a database) as is typically done with mtDNA results($fazCExample Y-STR Haplotype*Core US Haplotype DYS19  14 DYS389I  13 DYS389II  29 DYS390  24 DYS391  11 DYS392  14 DYS393  13 DYS385 a/b  11,15 DYS438  12 DYS439  13 ZZf f f f f f f f f f Matches by Databases 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 3,561ZZZZZZZZZZZ  ,Z G{D www.YHRD.org |E9Frequency Estimate Calculations Using the Counting Method:  }FbWhen there is no match with the counting method& 22 ~G#The Meaning of a Y-Chromosome Match$$ Conservative statement for a match report: The Y-STR profile of the crime sample matches the Y-STR profile of the suspect (at xxx number of loci examined). Therefore, we cannot exclude the suspect as being the donor of the crime sample. In addition, we cannot exclude all patrilineal related male relatives and an unknown number of unrelated males as being the donor of the crime sample. HZ)W&fH(Difficult Questions& Which database(s) should be used for Y-STR profile frequency estimate determination? Are any of the current forensic Y-STR databases truly adequate for reliable estimations of Y-STR haplotype frequencies? Some individuals share identical Y-STR haplotypes due to recurrent mutations, not relatedness& Is the database a random collection reflecting Y-STR haplotype frequencies of the population? Is the Y-STR haplotype frequency relevant for the population of the suspect?6 Uy  I Conclusions from Peter de KnijffA haplotype frequency taken from any Y-STR database should not be reported or seen as a random match probability Because all male relatives have the same haplotype Males can share haplotypes without being related (rfrfJ/What Peter de Knijff Reports with a Y-STR Match00YThe Y-STR profile of the stain matches with the suspect. Therefore, the suspect cannot be excluded as the donor of the stain. On the basis of this DNA evidence, I can also not exclude all paternally related male relatives of the suspect as possible donors of this stain. In addition, an unknown number of males from the same region cannot be excluded. A more accurate answer can only be obtained if (1) we have detailed knowledge of the population structure of the region of interest, (2) the Y-STR frequencies therein are known, and (3) we have knowledge about the family structure of the suspect.BZZI1B=Can Y-STR results be combined with autosomal STR information?>> Still subject to some debate among experts (most say  yes ) Problem of different inheritance modes Multiply random match probability from the autosomal STR profile obtained with the upper bound confidence limit from the Y-STR haplotype frequency estimate~'International Forensic Y-User Workshops(( Next meeting (5th): Sept 26-30, 2006 (Innsbruck, Austria)  will also cover mtDNA 1st  Berlin, Germany June 1996 2nd  Berlin, Germany June 2000 3rd  Porto, Portugal Nov 2002 4th  Berlin, Germany Nov 2004~+Mitochondrial DNA (mtDNA)+Comparison of Nuclear and Mitochondrial DNA,,7Identifying the Romanov Remains (the Last Russian Czar) 8 !Control Region (16024-576)* ^1,122 nucleotide positions Typically only 610 bases examined (HVI: 16024-16365; HVII: 73-340)8>!*!'Process for Evaluation of mtDNA Samples((#Differences from Reference SequenceChallenges with mtDNAData Interpretation Heteroplasmy Sample mixtures (currently not possible) DNA Database Sizes Similar issues to Y-STRs but takes longer to generate mtDNA data than Y-STR haplotypes DNA Database Quality`6W6W'Sequence Heteroplasmy at Position 16093(( Disadvantages to SequencingExpensive Primarily due to intensive labor in data analysis Error possibilities with more data to review Most information is not used N 2K 2KfAdvantages to Screening MethodsRapid results Aids in exclusion of non-matching samples Less labor intensive Usually less expensive Permits more labs to get involved in mtDNA7LINEAR ARRAY mtDNA Typing Strips: New Screening Method88 0A Common Use of mtDNA is for Hair Shaft Analysis 1  =Human hair shafts contain very little DNA but because mtDNA is in higher copy number it can often be recovered and successfully analyzed Melanin found in hair is a PCR inhibitor Important Publications: Wilson, M.R., et al. (1995) Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18(4): 662-669. Tissue grinding method described by FBI Lab Melton et al. (2005) Forensic mitochondrial DNA analysis of 691 casework hairs. J. Forensic Sci. 50(1): 73-80. Obtained a full or partial mtDNA profile for >92% of hairs testedZZZ,ZpZBZ,bB  ,B RRPCR Product Size Reduction Improves Recovery of STR Information from Telogen HairsSSfmtDNA and miniSTRsDue to the higher copy number, mtDNA will still have a role in many highly degraded DNA scenarios or where limited biological material is present, such as hair shafts. However, miniSTRs will most likely extend the range of cases where highly informative STR data can be obtained :THANK YOU FOR YOUR ATTENTION&  Thank you for attending and participating in this Advanced Topics in STR DNA Analysis Workshop Feel free to contact us if you have further questions: John Butler (NIST): john.butler@nist.gov Bruce McCord (FIU): mccordb@fiu.edu2ZNZN*B 0/    0 `X(  X X NǴ qq (   n*  S**SS}} X N( qq  (   p*  S**SS}}d X c $ ?l   4 X Nϴ qq  k   RClick to edit Master text styles Second level Third level Fourth level Fifth level!     S  X TtQ qq '    n*  S**SS}}  X T T qq  '   p*  S**SS}}H X 0lZ ? 3380___PPT10.`M  QI@(  >  NLbqq ]   JAAFS 2006 Workshop (Butler and McCord) Advanced Topics in STR DNA Analysis$K0J  S**SS}}  N0fqq +  rFebruary 20, 2006 &0  S**SS}}Q  Toqq  ]1   5http://www.cstl.nist.gov/biotech/strbase/training.htm$605  .S**SS}}  T\qq  w +1  ^* S**SS}}H  0lZ ? 3380___PPT10.Z 0 p\j(  \d \ c $Xm    \ s *ڴ X k   T@Figure 9.2 Schematic illustrating the types of autosomal or Y-STR profiles that might be observed with sexual assault evidence where mixtures of high amounts of female DNA may mask the STR profile of the perpetrator. Y-STR testing permits isolation of the male component without having to perform a differential lysis. H \ 0lZ ? 3380___PPT10. %0   0 @(  d  c $Xm     s *d X k   *Figure 9.5 Schematic illustration of how multiple PCR primer binding sites give rise to multi-copy PCR products for (A) DYS385 a/b and (B) DYS389I/II. Arrows represent either forward  F or reverse  R primers. In the case of DYS385 a/b, the entire region around the STR repeat is duplicated and spaced about 40,775 bp apart on the long arm of the Y-chromosome. Thus, amplification with a single set of primers gives rise to one peak if the  a repeat region is equal in size to the  b repeat region or separate peaks if  a and  b differ in length. DYS389 possess two primary repeat regions that are flanked on one side by a similar sequence. Widely used forward primers bind adjacent to both repeats generating amplicons that differ in size by ~120 bp. Note that DYS389II is inclusive of the DYS389I repeat region and therefore some analyses subtract DYS389II-DYS389I repeats.,<H  0lZ ? 3380___PPT10.; 0 91<(  <d < c $Xm   % < s *t X k   Figure 9.1 Illustration of inheritance patterns from recombining autosomal genetic markers and the lineage markers from the Y-chromosome and mitochondrial DNA.H < 0lZ ? 3380___PPT10.~ 0 xp D(  Dd D c $Xm   d D s * X k   Figure 9.3 An example pedigree showing patrilineal inheritance where all shaded males have the same Y-chromosome barring any mutations. To help identify the person in question, any of the other males with the same patrilineage could provide a reference sample to assist in a missing persons investigation, mass disaster victim identification, or deficient paternity test (boxed region) where the father is deceased or not available for testing.' H D 0lZ ? 3380___PPT10.w@\ 0 0 Ll(  Ld L c $Xl    L s *u X k   VBFigure 2.3 The human genome contained in every cell consists of 23 pairs of chromosomes and a small circular genome known as mitochondrial DNA. Chromosomes 1-22 are numbered according to their relative size and occur in single copy pairs within a cell s nucleus with one copy being inherited from one s mother and the other copy coming from one s father. Sex-chromosomes are either X,Y for males or X,X for females. Mitochondrial DNA is inherited only from one s mother and is located in the mitochondria with hundreds of copies per cell. Together the nuclear DNA material amounts to over 3 billion base pairs (bp) while mitochondrial DNA is only about 16,569 bp in length.H L 0lZ ? 3380___PPT10.O@ 0  d(  dd d c $Xl    d s *X k   TFigure 9.10 Ancestry of Thomas Jefferson and Eston Hemings male lines. The shaded boxes represent the samples tested by Foster et al. (1998) in their Jefferson Y chromosome study. A male descendant of Eston Hemings, son of Thomas Jefferson s slave Sally Hemings, was found to have a Y chromosome haplotype that matched male descendants of Field Jefferson, President Thomas Jefferson s uncle. A male descendant of Thomas Woodson, claimed by some to be descended from Jefferson, had a different Y haplotype and therefore could not have been a Jefferson.$+H d 0lZ ? 3380___PPT10.MG 0  l(  ld l c $Xl    l s *X k   |hFigure 10.1 Schematic showing the circular mitochondrial DNA genome (mtGenome). The heavy (H) strand is represented by the outside line and contains a higher number of C-G residues than the light (L) strand. The 37 RNA and protein coding gene regions are abbreviated around the mtGenome next to the strand from which they are synthesized (see Table 10.2). Most forensic mtDNA analyses presently examine only HV1 and HV2 in the non-coding control region or displacement loop shown at the top of the figure. Due to insertions and deletions that exist around the mtGenome in different individuals, it is not 16,569 bp.H l 0lZ ? 3380___PPT10.F 0   (  d  c $Xl     s * %X k   TFigure 10.4 Process for evaluation of mtDNA samples. The evidence or question (Q) sample may come from a crime scene or a mass disaster. The reference or known (K) sample may be a maternal relative or the suspect in a criminal investigation. In a criminal investigation, the victim may also be tested and compared to the Q and K results. T UH  0lZ ? 3380___PPT10.b 0 jb (  d  c $Xl   V  s *5X k   Figure 10.9 (A) Sequence heteroplasmy at position 16093 possessing both C and T nucleotides compared to (B) the same region (positions 16086-16101) on a different sample containing only a T at position 16093.H  0lZ ? 3380___PPT10.@ 4 0  "(  d  c $Xl   ~  s *;X k   Figure 10.10 (A) Results schematically displayed of a known (K) reference and a question (Q) sample that do not match one another using the Roche LINEAR ARRAY mtDNA HVI/HVII Region-Sequence Typing Strips. (B) Types are reported as a string of numbers representing the LINEAR ARRAY probe results. Failure of the PCR product to bind to a probe region (e.g., HVIE in sample Q) is referred to as a  blank , is reported as a zero in the string of numbers, and is due to polymorphisms in the sample near the probe site that disrupt hybridization. Weak signals such as indicated by the arrow for 189 in sample Q are also due to a closely spaced polymorphism that disrupts full hybridization of the PCR product to the sequence-specific probe present on the LINEAR ARRAY. H  0lZ ? 3380___PPT10.D 0  5(  d  c $Xl     s *@X k    Figure 10.6H  0lZ ? 33 0  5(  d  c $Xl     s *PX k    Figure 10.6H  0lZ ? 33 0 yq  (  d  c $Xl   e  s *UX k   Figure 10.8 (A) Comparison of sequence alignments for hypothetical Q and K samples with (B) conversion to the revised Cambridge Reference Sequence (rCRS) differences for reporting purposes.&H  0lZ ? 3380___PPT10. 0  d5(  dd d c $Xl    d s *]X k    Figure 10.6H d 0lZ ? 33rt |Z E }>> D j\  vcPQmXZ>"Root EntrydO)v*x PicturesCurrent User58SummaryInformation(T<      !"#$%&:./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~  !"#$%&'()*+,-./01234/www.familytreedna.com/index.htmlKhttp://www.relativegenetics.com/servlet/moonshine/goto?page_url=/index.jspurl=/index.jsp _N JmButlerJmButlerlerButler    ;w>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuv ~yz{|}