NEAFS2006 YmtDNA MtDNA3 - STRBase Introduction

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NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Y-Chromosome and Mitochondrial DNA AnalysismitochondrialDNANEAFS 2006 WorkshopRye Brook, NYNovember 1, 2006Dr. John M. ButlerDr. Michael D. lA Brief Sidestep mtDNA as a genetic tool “mitogenomics” The lack of apparent recombination, and highmutation rate make mtDNA an excellent tool forstudying human evolution. Some of these insights have also been useful forthe mtDNA forensic scientist.Methods for Measuring mtDNAVariation Low-resolution RFLP (1980s) High-resolution RFLP (1990s) Sequence analysis of HV1 and HV2 withincontrol region (1991-present) Sequence analysis of complete mtDNA ch/strbase/training.htm1

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006RFLP AnalysisHae IIImtDNA #1GGCCGGCC(800 bp)GGCCGGCC(1000 bp)mtDNA #2GGCCAGCCGGCC(loss of restriction site)(1800 bp)(GGTC)mtDNA #3GGCCGGCCGGCCGGCC(gain of restriction site)(800 bp)(600 bp)(400 bp)RFLP AnalysisDNALadder #1#2#3mtDNA #1GGCCGGCCGGCC2000(800 bp)(1000 bp)1000mtDNA #2800GGCCAGCCGGCC400(1800 bp)200(GGTC)mtDNA #3GGCCGGCC(800 bp)GGCC(600 bp)100GGCC(400 bp)Gel ImagemtDNA as a Genetic MarkerCann et al. 1987High res. RFLP analysis of 134mtDNA types from 148 individuals 370 different restriction sitesper individual.“Out of training.htm2

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006mtDNA as a Genetic MarkerControl Region SequenceAnalysis of 189 individualsVigilant et al. 1991mtDNA as a Genetic Marker Templeton (1992) Science – Found phylogenetictrees that were more parsimonious than Vigilantet al. AND these trees did not suggest an “Out ofAfrican” origin. More sequence data and better tree-buildingmethods confirmed the OOA hypothesis (Pennyet al. 1995; Watson et al. 1997)mtDNA as a Genetic MarkerIngman et al. (2000)53 entire genomesequences from diverseglobal populations.Confirmation for ing.htm3

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006mtDNA as a Genetic Marker RFLP variation has revealed continent-specificpolymorphisms for classifying mtDNAs. Haplotype – the mtDNA sequence variationswithin an individual (e.g. your HV1/HV2 type). Haplogroup – a group of related haplotypes.These form monophyletic clades on aphylogenetic tree.mtDNA HaplogroupsEach haplogroupcluster is defined by aset of specific, sharedpolymorphisms.In this cluster, all individualsbelonging to the African L2haplogroup share a set of 7SNPs in the coding region.Herrnstadt et al. AJHG (2002)mtDNA HaplogroupsIn this cluster, all individualsbelonging to the African L2bsub-haplogroup share a set of 17SNPs in the coding region.Herrnstadt et al. AJHG ining.htm4

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006mtDNA HaplogroupsIn this cluster, all individualsbelonging to the African L2asub-haplogroup share a set of 8SNPs in the coding region.Herrnstadt et al. AJHG (2002)Human Migration ModelmtDNA Haplogroups (HV1/HV2) J - 16069 C-T 16126 T-C 73 A-G 295 C-T T - 16126 T-C 16294 C-T 73 A-G V - 16298 T-C 72 T-C L3e3 - 16223 C-T 16265 A-C 73 A-G 150 C-T 195 T-CGenerally, very good concordance betweenCR and coding haplogroupsMacaulay et al. (1999) AJHG 64: 232-249Allard et al. (2002) JFS 47: 1215-1223Brandstatter et al. (2004) IJLM 118: aining.htm5

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Tools for mtDNA ScreeningDisadvantages to Sequencing Expensive– Primarily due to intensive labor in dataanalysis Error potential with more data to review Most information is not usedReview forward and reverse sequences across 610 bases only to report 263G, 315.1CMost common type: foundin 7% of Caucasians Advantages to Screening Methods Rapid resultsAids in exclusion of non-matching samplesLess labor intensiveUsually less expensivePermits more labs to get involved inmtDNASequencing is necessary to certify that every positionmatches between a question and a known sample.Screening assays are essentially a presumptive testprior to final confirmatory DNA e/training.htm6

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Methodologies for SNP TypingHigh-tech SNaPshot(minisequencing) Luminex 100 allele-specifichybridization Pyrosequencing TaqMan Primer extension with timeof-flight mass spectrometry TagArray (SNPstream UHT) Affymetrix hybridization chipLow tech Reverse dot blot(LINEARARRAYs) PCR-RFLP Allele-specific PCRSee Budowle et al. (2004) Forensic Sci. Rev. 16:21-36for a review of some SNP typing technologiesMito “Strips” Roche Applied Science (Indianapolis, IN)has released a mtDNA typing kit. LINEAR ARRAY Mitochondrial DNAHVI/HVII Region-Sequence Typing Kit Cat. No. 03 527 867 001 Cost 1500 for 50 reactions NIST was involved in beta-testing andperformed a population study with theseLINEAR ARRAYsPrevious Publications on mtDNATyping Assays with SSO Probes (dotblot, reverse dot blot, linear arrays) Stoneking et al. (1991) Population variation of human mtDNA control regionsequences detected by enzymatic amplification and sequence-specificoligonucleotide probes. Am. J. Hum. Genet. 48:370-382Skowasch, K., et al. (1994) Development of PCR-based reverse dot-blot typingsystem for the control region of mtDNA. Proceedings of the Fifth InternationalSymposium on Human Identification, Madison, WI: Promega, p. 127.Comas, D., et al. (1999) Eur. J. Hum. Genet. 7:459-468Terry MeltonCalloway, C.D., et al. (2000) Am. J. Hum. Genet. 66:1384-1397populationReynolds, R., et al. (2000) J. Forensic Sci. 45(6):1210-1231studies Gabriel, M.N., et al. (2001) Croatian Medical Journal 42(3):328-335Gabriel, M.N., et al. (2003) Croatian Medical Journal 44(3):293-298Calloway, C., et al. (2003) Validation of the LINEAR ARRAY Mitochondrial DNAHVI/HVII Region-Sequence Typing kit. Proceedings of the 14th InternationalSymposium on Human Identification.Calloway, C., et al. (2003) Applications of the LINEAR ARRAY Mitochondrial DNAHVI/HVII Region-Sequence Typing kit. Proceedings of the 14th InternationalSymposium on Human Identification.Kline, M.C., et al. (2005) J. Forensic Sci. ase/training.htm7

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Am. J. Hum. Genet. 48:370-382, 1991Population Variation of Human mtDNA Control RegionSequences Detected by Enzymatic Amplification andSequence-specific Oligonucleotide ProbesMark Stoneking, Dennis Hedgecock, Russell G. Higuchi, Linda Vigilant, and Henry A. ErlichDepartment of Human Genetics, Cetus Corporation, Emeryville, CA; Bodega Marine Laboratory, University of California, Bodega Bay, CA;and Division of Biochemistry and Molecular Biology, University of California, BerkeleyDot blot assayControl RegionHVIAIA1IA2IA31612616129HVIIBC DIB1IB2IB3IC1IC2IC3ID1ID216217 16304 1636216223 16311ABCIIA1IIA2IIB1IIB2IIB3IIC1IIC2IIC3731461527 SNP sites23 probesacross 9 regionsD EIID1 IIE1IID2 IIE214 SNPs195 247 309.11997 SNP sitesHVIISSO 7ABCIIA1IIA2IIB1IIB2IIB373146150152TTTTTTT309.110 SNP sitesDetection ChemistrySaiki, R.K., et al. (1989) Genetic analysis of amplified DNAwith immobilized sequence-specific oligonucleotide probes.Proc. Natl. Acad. Sci. USA ne 6026216029Biotin-labeled PCR productBiotin - .GAATATTGTACGGTACCAT .-3’CTTATAACATGCCATGGTA-5’ProbeNylon raining.htm8

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNAHVIIA160931 2 31 2RefHVIIIDICIIAIE1 21 2 3 41010 probeprobe regionsregionsIICIIB1 2IID 1891 2 4 51 2 3November 1, 20061 2 3 4 5 6 71 2311 2probesprobesCRS16093A T T T C. . C . .16093 116093 2IA 1IA 2IA 316126T G T A. . C . . . .16129C G G T. . . . A . .IC 1IC 2IC 3IC 4A.ID 1ID 216362C G T C C. . C . .IE 1IE 2IE 31627016278C A C T A G G A T A C C A. . . . . . . . . . T . . . T . . . . . . . . . .16304G T A. C . . . . .C.16309 16311A T A G T A C. . . . . . . . . . C . . . G . . . .IIA 1IIA 273G T A G T. . G . .IIB 1IIB 2IIB 3IIB 4IIB 5IIB 6IIB 7C.146C T C. C . . . C . . . . . C .IIC 1IIC 2IIC 4IIC 5G.189A A C. . . . . G .IID 1IID 2247T T G A A. . A . .189 1189 2189G A A C A. . G . .1818 SNPsSNPsA.150 152T C C T A. . . . . . . C . . . C . T . . . T . C . T . C .T.A.T.195C T T. C . C . . .A.A.198 200C T A A. . . .T . . . . G .A.Roche (F15975)FBI A1 (L15997)GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCTCTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACAGACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT1603016040160501606016070Hypervariable Region I16024-16365342 bp examined16080Roche IA16093 C16126 C A 16129ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAATTGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA160901610016110161201613016140HVI C-stretchACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTATGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT161501616016170161801619016200CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACCGTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG162101622016230162401625016260Roche ICRoche IETTCG CCCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGCGGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG162701628016290163001631016320SSO 2Only 9 sitesexaminedRoche IDCHV1CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCCGTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG163301634016350163601637016380TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCTAGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA163901640016410164201643016440Roche (R16418)FBI B1 (H16391)Roche (F15)FBI C1 (L048)GATCACAGGT CTATCACCCT ATTAACCACT CACGGGAGCT CTCCATGCAT TTGGTATTTTCTAGTGTCCA GATAGTGGGA TAATTGGTGA GTGCCCTCGA GAGGTACGTA AACCATAAAA10Roche IIA732030405060HV2GCGTCTGGGGG GTATGCACGC GATAGCATTG CGAGACGCTG GAGCCGGAGC ACCCTATGTCGCAGACCCCC CATACGTGCG CTATCGTAAC GCTCTGCGAC CTCGGCCTCG TGGGATACAG708090100110140150160170180Roche IIC 198195 C T G 200189 GACAGGCGAAC ATACTTACTA AAGTGTGTTA ATTAATTAAT GCTTGTAGGA CATAATAATATGTCCGCTTG TATGAATGAT TTCACACAAT TAATTAATTA CGAACATCCT GTATTATTAT190200210220230240Roche IIDAACAATTGAAT GTCTGCACAG CCACTTTCCA CACAGACATC ATAACAAAAA ATTTCCACCATGTTAACTTA CAGACGTGTC GGTGAAAGGT GTGTCTGTAG TATTGTTTTT TAAAGGTGGT247250260270280290300HV2 C-stretchHV2AACCCCCCCT CCCCCGCTTC TGGCCACAGC ACTTAAACAC ATCTCTGCCA AACCCCAAAATTGGGGGGGA GGGGGCGAAG ACCGGTGTCG TGAATTTGTG TAGAGACGGT TTGGGGTTTT31032033034035073-340268 bp examined120Roche IIBC 150 T C 152GCAGTATCTG TCTTTGATTC CTGCCTCATC CTATTATTTA TCGCACCTAC GTTCAATATTCGTCATAGAC AGAAACTAAG GACGGAGTAG GATAATAAAT AGCGTGGATG CAAGTTATAA146130Hypervariable Region IISSO Probes73146150152189195198200247Only 9 sitesexamined360ACAAAGAACC CTAACACCAG CCTAACCAGA TTTCAAATTT TATCTTTTGG CGGTATGCACTGTTTCTTGG GATTGTGGTC GGATTGGTCT AAAGTTTAAA ATAGAAAACC GCCATACGTG370380390400410FBI D1 (H408)420Roche (RR429)TTTTAACAGT CACCCCCCAA CTAACACATT ATTTTCCCCT CCCACTCCCA TACTACTAATAAAATTGTCA GTGGGGGGTT GATTGTGTAA TAAAAGGGGA GGGTGAGGGT ov/biotech/strbase/training.htm9

November 1, 2006NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNAData Interpretation for LINEAR ARRAYsAnalysis of probe resultsis still 512Typing results from 50 ng of each PCR productMargaret Kline, NIST“Blank” Calls on LINEAR ARRAYsNIST observed 640 “blanks” (9.6% of calls) on 346 different individuals (52% of samples typed).*Different individuals typing as a blank for the same probe region could have different substitutions but for the purposes of dataanalysis the blanks are considered to represent the same variant (see Melton et al. (2001) J. Forensic Sci. 46(1):46-52)SSO ProbeRegionNumberObservedFrequencyBudowle etal. (1999)Cau, AA, His16093233.5%HVIA335.0%3, 9, 3%HVIC7611.4%3, 20, 10%HVID335.0%9.0%7, 17, 4%HVIE60HVIIA30.5%0, 0, 0%HVIIB9614.4%16, 70,55%18.3%11, 47,13%5, 5, 18%HVIIC122HVIID426.3%18915222.8%PCR product fails to hybridize to anyprobe in region due to additionalpolymorphisms in the probe regionthat prevent hybridizationProbe Region HVIIBBlanks expectedbased on fullsequence analysisof 1393 individualsIIBIIBIIBIIBIIBIIBIIB1234567C.C.146T CC . .C . . .C .A.T.150 152C C T A. . . . . C . . C .T . . .T . C .T . C .T.Nucleotide positions 151 and 153 arecommon variants in African AmericansKline et al. (2005) JFSTyping frequencies for 666NIST population .01.71.82.73.54.27.7Summary of NIST PopulationTyping with Roche mtDNALINEAR ARRAYS 282 different types 185 were unique(occurred only once) 51 samples had “MostCommon Type”“Most Common Type” evaluated furtherwith mtDNA coding region SNP se/training.htm10

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Comparison of Other U.S. Population Datawith SSO ProbesPopulationN#typesdiversityMost Common TypeMCT can 01111.7%Total22825020.998111111117.2%Melton et al. (2001) J. Forensic Sci. 46(1): 46-528 regions, 21 probes, 13 SNPsPopulationN#typesdiversityMost Common TypeMCT n 12011116.4%Total6662820.98511111111117.7%Kline et al. (2003) NIST population study10 regions, 31 probes, 18 SNPsHV1 Null AllelesAHVII Array Locus(rCRS position)16093AfricanAmerican Hispanic Caucasian887Haplogroup AssociatedPolymorphisms--Percentage of Null Allelesfrom Haplogroup Polymorphisms--HVIA(16126; 16124)242716124C - L3b and L3d27/33 (82%)HVIC(16304; 16309; 16311)28381016320T - L3e216290T and 16319A - A (Asian)69/76 (91%)HVID(16362)293216360T - L1c29/34 (85%)HVIE(16270; 16278)4011816270T and 16278T - L1b16264T - L3e416265T - L3e316265C - L1c233/59 (56%)1296234225158/225 (70%)totalCoble et al. (in review)HV2 Null AllelesBHVII Array Locus(rCRS position)HVIIA(73)AfricanAmerican Hispanic Caucasian102Haplogroup AssociatedPolymorphisms72T-C - preVPercentage of Null Allelesfrom Haplogroup Polymorphisms1/3 (33%)HVIIB(146; 150; 152)463615151C-T - L1c143G-A - L2a*153A-G - A2; X*88/97 (91%)HVIIC(189; 195; 198; 200 )661937186 C-A - L1c185G-T - L1b189A-C - L2b/c185G-A; 189A-G; and 200A-G - L3e*194C-T - D*/D4b2199T-C and 204T-C - I78/122 (64%)HVIID(247)5277249A-del - CZ242C-T - J1*250T-C - I2*32/39 (82%)HVIE(189)1031832182C-T and 195T-C L1*/L2*185G-T - L1b195T-C and 198C-T - L2a*189A-C - L2b/c185G-A and 200A-G - L3e1a185G-A - J1*194C-T - D*/D4b2123/153 (80%)22110093414322/414 (78%)totalCoble et al. (in aining.htm11

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Sequencing – IncreasedDiscrimination# timeshaplotypeobserved LINEAR D% DC# HT0.986942.19%281HV1HV1 HV2control otal666Coble et al. (in review)SNP Typing InstrumentationMulti-Color Capillary Electrophoresis(ABI 310 or 3100)PCRPCR&& primerprimer extensionextensionTaqManTaqManABI 7000 SDSSNP Extension Primer Design Must anneal to DNA template with 3’end ofprimer next to SNP site Can anneal to either top strand or bottom strand Should have uniform annealing temperature (bylengthening 5’end of SNP primer) Should not form significant hairpins or dimerswith other SNP or PCR h/strbase/training.htm12

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Early Multiplex SNP Detection Work16069 1612916189 162241631173 146152 195247 309.1523 525Variable poly(T) 5’tails to spread pecific Primer ExtensionSNP Primer is extended by one base unitABI PRISM SNaPshot Multiplex System“tail” used to vary electrophoretic mobilityOligonucleotide primer 18-28 bases5’3’CGTFluorescentlylabeled ddNTPs polymeraseAGPCR Amplified DNA TemplateProducts can be electrophoretically separated on an ABI 310, 3100Allele-Specific Primer ExtensionThe use of “tailed” SNP primers allows formultiplexing in the SNaPshot assaySequences for 11 SNP 5025/5420/5821/62Template binding sequence – blackTailed sequence for fragment separation - ng.htm13

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Allele-Specific Primer Extension28G20A36G20 nucleotides52C 52TSNaPshot CEPH Control ReactionddA36 nucleotidesTTTT60C44T28ATTTTTTTTddG44 nucleotidesTTTTTTTTTTTTTTTTddT60 nucleotidesPriming siteddCProtocol with SNaPshot “Kit”AmplificationGenomicDNA sample(Multiplex)PCR2.5 hoursPrimerExtensionAdd SNPprimer(s) andSNaPshot mixAnalysisSNP Extension(cycle sequencing)1 hourSample prep for310/3100Run on ABI310/3100Add GS120 LIZsize standardUse E5 filter (5-dye) andPOP4 standard conditions24 min on 3100ExoSAPDigestion1 hourSAPtreatment1 hourData Analysis(GeneScan)Type sample(Genotyper 3.7)Or GeneMapperUse of Haplogroup Defining mtSNPsIdentified coding region SNP to classify the 9major Western European haplogroups (plus2 strbase/training.htm14

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Use of Haplogroup Defining mtSNPsBrandstatter et al. C15904T16 mtSNPs run in twoSNaPshot 8plex reactionsUse of Haplogroup Defining mtSNPsHaplogroups Defined by Control Region mtSNPs inSWGDAM Caucasian Samples (n 1771)Allard et al. (2002) J. Forensic Sci. 47(6):1215-1223 I (2%): 16223T, 199C, 204C,250C H (46%): 73A U (15.6%): 16270T V (1.9%): 16298C, 72C T (10.5%): 16126C, 16294T J (10%): 16069T, 16126C,295T K (8.9%): 16224C, 16311C W (1.9%): 16223T, 189G,195C, 204C, 207A X (1.6%): 16189C, 16223T,16278T, 195C M (1.9%): 16223T, 16298CIf a G is observed at 8251, then the sample can be classified as a member ofhaplogroup X so the following control region SNPs should be expected: 16189C,16223T, 16278T, 195CUse of Haplogroup Defining mtSNPsAdvantagesSensitive – 1 pg genomic DNAShort amplicons – degraded DNAMultiplexed PCR – conserves templatePOD – 88.6% among 277 unrelated Austrian CaucasiansDisadvantagesNo “kit” – need to order, validate primers.Probability of random match among Caucasians is ning.htm15

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Emerging mtDNA technologiesmtDNA genome sequencing forincreased discriminationmtDNA Population DistributionCaucasians (n 1665)Number of HV1/HV2 0.050.060.070.08Percentage of Population with a Particular HV1/HV2 TypeCoble et al. (2004)mtDNA Population DistributionCaucasians (n 1665)Number of HV1/HV2 Types900Over one-half are “unique”800700600500400300A small number are 08Percentage of Population with a Particular HV1/HV2 TypeCoble et al. ining.htm16

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Framing the Problem The greatest limitation for mtDNA testing lieswith the small number of common types forwhich the power of discrimination is low. 20% of the time, the Forensic Scientistencounters a HV1/HV2 type that occurs atgreater than 0.5% of the population. In database or mass fatality comparisons:multiple hits will occur for these common types.A Case Example September 15, 1943 - B17F Bomberreturning from a mission to Port Moresby,New GuineaA Case Example The plane crashes in the Owen StanleyMountain range due to “adverse weather.” Subsequent searches proved negative. 11 crewmen declared non-recoverable onJuly 22, ning.htm17

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006A Case Example October 9, 1992 - A private companyhelicopter discovers crash site. mtDNA testing reveals that 3/11 crewmenshare the same HV type (263 A-G, 315.1C). Further VR testing could distinguish 1 ofthe 3 crewmen (16519 T-C). However, 2crewmen still matched.A Case Example Partial dental records were used toassociate 3 teeth among the 2 crewmenmatching in the CR. One L femur could not be associated witheither crewmen, and was buried in a gravecontaining group remainsStrategy for SNP Identification Sequence the entire genome of unrelatedindividuals sharing common HV1/HV2types in the Caucasian population (focuson 18 of 22 common types that occur at afrequency of 0.5% or training.htm18

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Ethical Considerations 265 characterized diseases associatedwith mtDNA mutations in the coding region(Mitomap – www.mitomap.org) To avoid having forensic testing fromevolving into genetic counseling, wedecided to focus on neutral SNPs in themtGenome.SNPs for Discrimination Non-coding sites in the control region(outside of HV1/HV2). Non-coding “spacer” regions throughoutthe mtGenome. Silent mutations in protein coding genes.SNPs for Discrimination Practical application – A set of SNP sitesthat can be rapidly assayed to providemaximal discrimination. Avoids further sequencing. Allele Specific Primer Extension – smallamplicons, multiplexed - can conservetemplate, run on standard trbase/training.htm19

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Common mtDNA HaplogroupsLength Variation in HV2 C-stretch – ignored (Stewart et al. (2001)Common mtDNA Haplogroups241 total genomes from 18 common HV1/HV2 types( 14% of the total database)Strategies for Whole mtGenome Analysis58 PCR rxn24 PCR rxn18 PCR rxn116 seq rxn48 seq rxn36 seq rxnLevin et al. (1999)Rieder et al. (1998)Ingman et al. (2000)Aldridge et al. (2003)12 PCR rxn32 PCR rxn15 PCR rxn95 seq rxn64 seq rxn47 seq rxnCoble et al. (2004)Maca-Meyer et al. (2001)Kong et al. ining.htm20

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Sequencing StrategymtDNAgenome12 Fragments(Levin et al. 1999)Construct Contigs(Sequencher)Each fragment issequenced F/RSeparation usingthe ABI 310096 well formatThe Nature of the SNPs Would the SNPs that resolve one group beuseful for resolving other closely relatedgroups?“Hot Spots”The Nature of the SNPs Are resolving SNPs slow and rare? Did theseSNPs arise once during the evolution of ahaplogroup?OR Are resolving SNPs “universally” fast hot spots,useful for all haplogroups (L, M, N)?OR . Are resolving SNPs a combination of the ing.htm21

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006263 A-G315.1 C13 SNPs14 types263 A-G315.1 C263 A-G315.1 .htm22

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006263 A-G315.1 CHaplogroup V(Reversion at 16298 CCSequence with HgVbackground appearsas a HgH (H1)H4 - rCRS 16263 ng.htm23

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006T1 – Low Resolution67% - not resolvedBrute-Force Sequencing Why not used information from theliterature? Prior to 1999, only a handful of wholegenome sequences in GenBank. Most ofthe mtDNA coding region data was fromRFLP studies (assays 20% of thegenome)mtGenomicsJan. 2006 - Kivisild et al.(277 complete – global)Oct. 2004 - Tanaka et al.(672 complete Japanese)May 2002 - Herrnstadt et al.(560 coding only)Jan. 2003 - Mishmar et al.(48 complete - global)Aug. 2001 - Maca-Myer et al. (42 complete - global)Dec. 2000 - Ingman et al. (53 complete - aining.htm24

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006mtDB - Human MitochondrialGenome Database Max Ingman (Uppsala University, Sweden) 1711 complete sequences and 839 codingregion sequences. 2550 coding region sequences.mtDB - Human MitochondrialGenome Database9380 G-A has only been observed in 11/2296 (0.48%)coding regions would not be a good candidate ifone was “trolling” the database for discriminating SNPsProblem – very few common types in global DBSummary 241 mtGenomes – 420 polymorphic sitesin the coding region. 32/241 (13%) – matched one or moreindividuals over the entire mtGenome(0/12 H5 individuals matched; 4/8 H7individuals matched). Homoplasies – common in raining.htm25

NEAFS Y-mtDNA Workshop (Butler and Coble)mtDNANovember 1, 2006Homoplasy – Parallel Substitutions3 basal polymorphisms thatdefine3 clusters“Red” mutation has occurredmultiple times on the treeSummary Percentage of sites that varied ranged from1.0% (16S rRNA) to 6.6% (non-coding regionsoutside of the control region). ATP Synthase 8 (4.8%) and ATP Synthase 6(3.7%) showed the greatest non-synonymousvariation in the protein coding s81194520421715715Total22363818827187637541538% 26

16390 16400 16410 16420 16430 16440 FBI A1 (L15997) Roche (F15975) HV1 16093 16126 16129 HVI C-stretch Roche IA Roche ID Roche IE Roche IC HV1 FBI B1 (H16391) Roche (R16418) Hypervariable Region I 16024-16365 342 bp examined SSO Probes 16093 16126 16129

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Advertise Monetize CPS 소개서 TNK CPS Introduction 매체소개서 Monetize Introduction About Us TNK Factory Introduction 회사소개서 DSP 소개서 TNK DSP Introduction 퍼포먼스 소개서 Performance Introduction 코드뱅크 소개서 Codebank Introduction TNK Factory는 안전하고 빠르며 쉬운 플랫폼입니다.

An Introduction to Modal Logic 2009 Formosan Summer School on Logic, Language, and Computation 29 June-10 July, 2009 ; 9 9 B . : The Agenda Introduction Basic Modal Logic Normal Systems of Modal Logic Meta-theorems of Normal Systems Variants of Modal Logic Conclusion ; 9 9 B . ; Introduction Let me tell you the story ; 9 9 B . Introduction Historical overview .

Partie 1 : Introduction et fonctions 1-1-1 Section 1 : Introduction Surveillance STEPS de l'OMS Section 1: Introduction Présentation générale Introduction Cette section constitue une introduction au Manuel de l'OMS pour la surveillance STEPS. Objectif L'objectif du Manuel est de proposer des lignes directrices et de fournir des

Example risk assessment for food preparation, cooking and service This example risk assessment applies to restaurants, cafés, sandwich bars, pubs, takeaways or hotel kitchens.