1 reasonable safeguards against contamination in mtdna testing, and some database issues dr....
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Reasonable Safeguards against Reasonable Safeguards against Contamination in mtDNA Testing, Contamination in mtDNA Testing, And Some Database IssuesAnd Some Database Issues
Dr. Frederika Kaestle (Depts of Dr. Frederika Kaestle (Depts of Anthropology and Biology, IU Anthropology and Biology, IU Bloomington) and Dr. Jason Eshleman Bloomington) and Dr. Jason Eshleman (Trace Genetics)(Trace Genetics)
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Ancient DNA and Forensic Ancient DNA and Forensic DNA AnalysisDNA Analysis
Similarities: Similarities: Template DNA unknownTemplate DNA unknown Often limited templateOften limited template Template often highly degradedTemplate often highly degraded Far from ideal sources for biological Far from ideal sources for biological
researchresearch
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Ancient DNA and Forensic Ancient DNA and Forensic DNA AnalysisDNA Analysis
Differences: Differences: Ancient DNA still largely limited to mtDNA Ancient DNA still largely limited to mtDNA
analysis.analysis. Ancient DNA is rarely searching for exact Ancient DNA is rarely searching for exact
match.match. Audience (justice system vs. academic Audience (justice system vs. academic
community) familiarity with genetics differ community) familiarity with genetics differ significantly.significantly.
Level of skepticism remains high with ancient Level of skepticism remains high with ancient DNA.DNA.
Hypothesis testing is overtHypothesis testing is overt
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Limitations of Ancient DNALimitations of Ancient DNA
Minimal template.Minimal template. PCR inhibitors co-extracted.PCR inhibitors co-extracted. Sources have often been heavily Sources have often been heavily
handled.handled. That property of mtDNA (high copy That property of mtDNA (high copy
number) that makes it possible to number) that makes it possible to analyze likewise makes contamination a analyze likewise makes contamination a more significant problem. more significant problem.
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Contamination SourcesContamination Sources
Lab-ware and reagents come ‘pre-Lab-ware and reagents come ‘pre-contaminated’ from the manufacturercontaminated’ from the manufacturer PCR tubes are particularly notorious PCR tubes are particularly notorious
(Schmidt et al. [1995] estimates high rate of (Schmidt et al. [1995] estimates high rate of mtDNA contamination in lab disposables)mtDNA contamination in lab disposables)
Taq (the enzyme that catalyzes PCR) has Taq (the enzyme that catalyzes PCR) has also been shown to be contaminated with also been shown to be contaminated with mtDNAmtDNA
Other reagents shown to be contaminated Other reagents shown to be contaminated include nucleotides, buffer, primersinclude nucleotides, buffer, primers
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Contamination SourcesContamination Sources
““Sample” surfaceSample” surface Contamination may have existed prior to sample Contamination may have existed prior to sample
(e.g. bone, hair) being deposited(e.g. bone, hair) being deposited Contamination may have occurred after deposition Contamination may have occurred after deposition
but before collectionbut before collection Contamination may have occurred during the Contamination may have occurred during the
collection of the samplecollection of the sample Contamination may have occurred during the Contamination may have occurred during the
storage and transport of the samplestorage and transport of the sample Contamination may have occurred at the laboratory Contamination may have occurred at the laboratory
after sample deliveryafter sample delivery
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Contamination SourcesContamination Sources
Carryover PCRCarryover PCR Contamination may occur due to residues from Contamination may occur due to residues from
previous PCR amplifications in the lab remaining on previous PCR amplifications in the lab remaining on lab ware, lab surfaces, lab clothing, lab equipment, lab ware, lab surfaces, lab clothing, lab equipment, lab airlab air
This is particularly problematic due to the high copy This is particularly problematic due to the high copy number of the PCR ampliconsnumber of the PCR amplicons
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Contamination SourcesContamination Sources
PeoplePeople With or without access to facilitiesWith or without access to facilities
Lab personnel shed DNA throughout the dayLab personnel shed DNA throughout the day Lab personnel carry DNA from others into the Lab personnel carry DNA from others into the
lab every day on the surfaces of their clothing lab every day on the surfaces of their clothing and bodyand body
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Identifying contaminationIdentifying contamination(or “how to have your paper rejected upon submission”)(or “how to have your paper rejected upon submission”)
Does sample match lab personnel?Does sample match lab personnel? But you can’t rule out everyone. Identifying But you can’t rule out everyone. Identifying
contamination requires some serendipity.contamination requires some serendipity. Understand negative controls are at times Understand negative controls are at times
insufficientinsufficient Low level in background can be mistaken for Low level in background can be mistaken for
sample. sample. Neg. control might not be contaminated, but sample Neg. control might not be contaminated, but sample
is (e.g. PCR tube)is (e.g. PCR tube) The sequence doesn’t “make sense.”The sequence doesn’t “make sense.”
But now we run the risk of only finding what we’re But now we run the risk of only finding what we’re looking for.looking for.
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What IS contamination?What IS contamination?
Unwanted DNAUnwanted DNA Why is it unwanted?Why is it unwanted?
Analyst did not intend to extract itAnalyst did not intend to extract it DNA is DNADNA is DNA
(It makes for a messy story? The study would be so (It makes for a messy story? The study would be so much nicer if we didn’t find it?)much nicer if we didn’t find it?)
Contamination does not answer the question at Contamination does not answer the question at hand.hand.
But what’s the question? But what’s the question?
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What Questions Can We What Questions Can We Answer?Answer?
Are two samples different?Are two samples different? What can we infer from this?What can we infer from this?
Not from the same source (but remember heteroplasmy, Not from the same source (but remember heteroplasmy, and possibility of contamination)and possibility of contamination)
Are two samples the same?Are two samples the same? What can we infer from this?What can we infer from this?
MIGHTMIGHT be from the same source (identity by descent, be from the same source (identity by descent, possibility of contamination)possibility of contamination)
Note that anthropologists often are asking Note that anthropologists often are asking questions about FREQUENCIES of mtDNA questions about FREQUENCIES of mtDNA types, not about single samplestypes, not about single samples Allows us to make population-level inferencesAllows us to make population-level inferences
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What IS contamination?What IS contamination?
Thus, one way to view contamination is Thus, one way to view contamination is as DNA that leads to a false inference *if* as DNA that leads to a false inference *if* we do not know out data are we do not know out data are compromised.compromised.
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So How Do We Detect So How Do We Detect Contamination?Contamination?
Protocols designed to detectProtocols designed to detect Negative controlsNegative controls
Reagent/Extraction blankReagent/Extraction blank Amp Negative/No Template ControlAmp Negative/No Template Control
Controls alert us to the presence of DNA in a tubeControls alert us to the presence of DNA in a tube We infer what this means vis a vis our samplesWe infer what this means vis a vis our samples
Comparison to sequences of probable contaminating Comparison to sequences of probable contaminating individualsindividuals Lab personnelLab personnel Excavators (evidence collection team)Excavators (evidence collection team) Museum Curators and Researchers (staff with access to Museum Curators and Researchers (staff with access to
evidence storage)evidence storage)
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Case Study: contamination Case Study: contamination and multiple inferencesand multiple inferences
1990- bones and teeth found in the 1990- bones and teeth found in the desertdesert
1996- bones identified as teenage boy, 1996- bones identified as teenage boy, missing in 1979missing in 1979
2002- mtDNA identifies match between 2002- mtDNA identifies match between bones and boy’s motherbones and boy’s mother
NEGATIVE CONTROLS CLEAN NEGATIVE CONTROLS CLEAN THROUGHOUTTHROUGHOUT
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Initial mtDNA resultsInitial mtDNA results
It is *possible* that the bone was really from the missing boy
If this is a match there is also contamination This is still merely a suggestive result as there are
at least four 2-source mixtures that can produce this result
Result requires confirmation
Mother: Mother: 16224, 16287, 16224, 16287, 1631116311
Bone: Bone: 16224T/C, 16224T/C, 16287C/T, 16311T/C16287C/T, 16311T/C
Conclusion: mixture, possible match
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Subsequent mtDNA Subsequent mtDNA ResultsResults
22ndnd extraction: ND extraction: ND (extraction failed)(extraction failed)
33rdrd extraction: extraction: 16085, 16085, 16111, 16223, 16257, 16261, 16111, 16223, 16257, 16261, 16286, 1631116286, 16311
44thth extraction: extraction: 16082A/C, 16082A/C, 16183A/C, 16189T/C, 16183A/C, 16189T/C, 16217T/C16217T/C, 16223, 16290, , 16223, 16290, 16291, 16319, 1636216291, 16319, 16362
55thth extraction: extraction: 16183A/c, 16183A/c, 16223, 16319G/a, 16325C/t, 16223, 16319G/a, 16325C/t, 16362T/C16362T/C
66thth extraction: extraction: 16183, 16183, 16187, 16189, 1621716187, 16189, 16217
77thth + 8 + 8thth extractions: extractions: 16224, 16287, 16224, 16287, 1631116311
Mother: Mother: 16224, 16287, 16224, 16287, 1631116311
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mtDNA ResultsmtDNA Results
Mother: Mother: 16224, 16287, 16224, 16287, 1631116311
Bone: Bone: 16224T/C, 16224T/C, 16287C/T, 16311T/C16287C/T, 16311T/C
77thth + 8 + 8thth extractions: extractions: 16224, 16287, 16224, 16287, 1631116311
Match confirmed!
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TimelineTimeline
22ndnd extraction (no result): 7/19/02 extraction (no result): 7/19/02
33rdrd extraction (no match): 8/6/02 extraction (no match): 8/6/02
44thth extraction (no match): 8/13/02 extraction (no match): 8/13/02
55thth extraction (no match): 8/22/02 extraction (no match): 8/22/02
66thth extraction (no match): 9/3/02 extraction (no match): 9/3/02
77thth extraction (matching): 9/10/02 extraction (matching): 9/10/02
88thth extraction (matching): 9/28/02 extraction (matching): 9/28/02
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TimelineTimeline
22ndnd extraction (no result): 7/19/02 extraction (no result): 7/19/02
33rdrd extraction (no match): 8/6/02 extraction (no match): 8/6/02
44thth extraction (no match): 8/13/02 extraction (no match): 8/13/02
55thth extraction (no match): 8/22/02 extraction (no match): 8/22/02
66thth extraction (no match): 9/3/02 extraction (no match): 9/3/02
77thth extraction (matching): 9/10/02 extraction (matching): 9/10/02
88thth extraction (matching): 9/28/02 extraction (matching): 9/28/02
Benchnotes reveal 9/10references and extractions performed at same time by same analyst.
Extractions 6, 7 and 8 performed on one bone sample.
New references: New references: 9/10/029/10/02 }
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Inferences?Inferences?
Possible that bones really are from missing Possible that bones really are from missing child.child.
Possible that errant handling contaminated Possible that errant handling contaminated bone with reference sample.bone with reference sample.
CERTAIN that improper lab practices were CERTAIN that improper lab practices were used.used.
Contamination existed, ignored when it did not Contamination existed, ignored when it did not fit with desired result.fit with desired result.
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Contamination Control Contamination Control Tools of the aDNA Trade Tools of the aDNA Trade
Keep it CleanKeep it Clean Surface decontamination of bone helps (Bouwman et al 2006)Surface decontamination of bone helps (Bouwman et al 2006) DNase I (Eshleman and Smith 2001) cleanup of reagents and tubesDNase I (Eshleman and Smith 2001) cleanup of reagents and tubes Positive Pressure HEPA-filtered air in the labPositive Pressure HEPA-filtered air in the lab Regular UV-irradiation of surfacesRegular UV-irradiation of surfaces Controlled and limited access to the labControlled and limited access to the lab Dedicated and disposable laboratory clothing and shoesDedicated and disposable laboratory clothing and shoes
Prevent CarryoverPrevent Carryover Uni-directional travel between extraction and PCR laboratoriesUni-directional travel between extraction and PCR laboratories Use of dUTP (Uracil) and pre-digestion of subsequent PCR reactionsUse of dUTP (Uracil) and pre-digestion of subsequent PCR reactions
(and ideally)(and ideally) Independent confirmation Independent confirmation (in temporally separate extraction/amplification (in temporally separate extraction/amplification
procedures and possibly at another laboratory)procedures and possibly at another laboratory) Split the samples first! Why analyze contamination twice? Split the samples first! Why analyze contamination twice?
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Kennewick Man Case Kennewick Man Case StudyStudy
3 independent ancient DNA laboratories, utilizing 3 independent ancient DNA laboratories, utilizing standard contamination controls standard contamination controls
3 independent samples of ancient Native American 3 independent samples of ancient Native American individual discovered near Kennewick, WAindividual discovered near Kennewick, WA
Results after multiple extraction and amplification Results after multiple extraction and amplification attempts (all negative controls clean):attempts (all negative controls clean): Lab 1: multiple failures at amplification, followed by sequence Lab 1: multiple failures at amplification, followed by sequence
identical to lab directoridentical to lab director Lab 2: multiple failures at amplification, followed by sequence Lab 2: multiple failures at amplification, followed by sequence
identical to student who had not been in the ancient DNA identical to student who had not been in the ancient DNA laboratory (or in town) for approximately 2 yearslaboratory (or in town) for approximately 2 years
Lab 3: multiple failures at amplification, followed by sequence Lab 3: multiple failures at amplification, followed by sequence identical to lab manager who had never entered the ancient identical to lab manager who had never entered the ancient DNA laboratoryDNA laboratory
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Neanderthal Case StudyNeanderthal Case Study
Neanderthal remains had been curated in European Neanderthal remains had been curated in European museum for several yearsmuseum for several years
Ancient DNA laboratory personnel extract DNA from Ancient DNA laboratory personnel extract DNA from tooth of a Neanderthal (39,900 years old), using tooth of a Neanderthal (39,900 years old), using standard precautionsstandard precautions
2 independent extractions performed2 independent extractions performed Both sequences identical to each otherBoth sequences identical to each other
Neanderthal?Neanderthal? Sequences compared to potential contaminating Sequences compared to potential contaminating
individualsindividuals Sequence identical to paleontologist who had studied remains Sequence identical to paleontologist who had studied remains
extensivelyextensively
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Reality CheckReality Check
There is no magic bulletThere is no magic bullet Contamination is a reality in DNA work.Contamination is a reality in DNA work. Negative controls are an indicator, not a Negative controls are an indicator, not a
solution, not a guarantee.solution, not a guarantee. Thinking clearly, asking the right Thinking clearly, asking the right
questions and posing the alternatives is questions and posing the alternatives is essential. essential.
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mtDNA DatabasemtDNA Database
Assuming you have mtDNA results, what Assuming you have mtDNA results, what do they mean?do they mean?
Previous presenters discussed many Previous presenters discussed many issues associated with the mtDNA issues associated with the mtDNA database, but I would like to concentrate database, but I would like to concentrate on an issue that has emerged from the on an issue that has emerged from the majority of anthropological genetic majority of anthropological genetic research on mtDNAresearch on mtDNA
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Relevant mtDNA BasicsRelevant mtDNA Basics
mtDNA inherited maternally, and does not mtDNA inherited maternally, and does not recombine – distribution of sequences is recombine – distribution of sequences is determined by movement of femalesdetermined by movement of females
mtDNA has very fast mutation rate compared mtDNA has very fast mutation rate compared to nuclear DNA – new mutations crop up all the to nuclear DNA – new mutations crop up all the timetime
This creates a situation in which new mtDNA This creates a situation in which new mtDNA lineages are very rare, and generally of limited lineages are very rare, and generally of limited distribution, and lineages in general are not distribution, and lineages in general are not randomly distributedrandomly distributed
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Relevant Anthropological Relevant Anthropological BasicsBasics
People do not move randomly on the People do not move randomly on the landscapelandscape Tend not to move large distancesTend not to move large distances Tend to follow family and friends, members of their Tend to follow family and friends, members of their
religion/language group/caste etc.religion/language group/caste etc. Tend to follow paths of ‘least resistance’ (valleys, Tend to follow paths of ‘least resistance’ (valleys,
rivers)rivers) Tend to follow jobs/game/other economically Tend to follow jobs/game/other economically
important resourcesimportant resources Are occasionally moved against their willAre occasionally moved against their will
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African American African American ExampleExample
Africans have highest level of mtDNA variation in the Africans have highest level of mtDNA variation in the world, and highest level of rare mtDNA sequencesworld, and highest level of rare mtDNA sequences
Slaves transported non-randomly to AmericasSlaves transported non-randomly to Americas South Carolina plantation owners grew mostly rice, so South Carolina plantation owners grew mostly rice, so
preferred slaves from West Africa who knew how to grow ricepreferred slaves from West Africa who knew how to grow rice Virginia plantation owners had to deal with malaria from Virginia plantation owners had to deal with malaria from
mosquitoes who thrived in surrounding swamps, so preferred mosquitoes who thrived in surrounding swamps, so preferred slaves from the “Gold Coast” of Africa who were resistant to slaves from the “Gold Coast” of Africa who were resistant to malariamalaria
Louisiana slave owners tended to purchase slaves from Louisiana slave owners tended to purchase slaves from Portuguese and French traders, who took slaves from AngolaPortuguese and French traders, who took slaves from Angola
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African American African American ExampleExample
During the “Great Migration” (1910-1930), large During the “Great Migration” (1910-1930), large numbers of African Americans moved out of numbers of African Americans moved out of the rural south (for better jobs in light of WWI the rural south (for better jobs in light of WWI and a boll weevil crop infestation)and a boll weevil crop infestation) Those from Mississippi, Alabama, Louisiana Those from Mississippi, Alabama, Louisiana
followed Miss R. north to large cites of Midwestfollowed Miss R. north to large cites of Midwest Those from Carolinas and Virginia followed the Those from Carolinas and Virginia followed the
coastline to D.C., Philly, NYC.coastline to D.C., Philly, NYC. But the majority of African Americans remain in But the majority of African Americans remain in
the SE even today.the SE even today.
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African American African American ExampleExample
In addition to actual population movement, the In addition to actual population movement, the genetic make-up of African American groups genetic make-up of African American groups across the US varies significantly in the level of across the US varies significantly in the level of admixture with non-Africansadmixture with non-Africans Level of admixture with European Americans is Level of admixture with European Americans is
higher in the West, and in large northern cities (likely higher in the West, and in large northern cities (likely due to different social mores)due to different social mores)
Level of admixture with Native Americans is higher Level of admixture with Native Americans is higher in the West and Southwest (probably due to a in the West and Southwest (probably due to a combination of social mores and the higher number combination of social mores and the higher number of Native Americans resident in the West)of Native Americans resident in the West)
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mtDNA DatabasemtDNA Database
Does the database take into account this Does the database take into account this regional variation in African American mtDNA regional variation in African American mtDNA sources?sources? NONO
Of ~1148 samples, approximately 800 are from HoustonOf ~1148 samples, approximately 800 are from Houston The samples overall are convenience samples from The samples overall are convenience samples from
“blood banks, paternity-testing laboratories, “blood banks, paternity-testing laboratories, laboratory personnel, clients in genetic-counseling laboratory personnel, clients in genetic-counseling centers, law-enforcement officers, and people centers, law-enforcement officers, and people charged with crimes” (NRC II (1996) supra note 5, charged with crimes” (NRC II (1996) supra note 5, at 30), and are thus in no way randomized with at 30), and are thus in no way randomized with regard to geographic origin or census data on the regard to geographic origin or census data on the distribution of African Americans in the US.distribution of African Americans in the US.
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mtDNA databasemtDNA database
The other subsets of the database suffer The other subsets of the database suffer from the same problem of non-random from the same problem of non-random sampling alsosampling also E.g. all of the Native American samples are E.g. all of the Native American samples are
Apache and Navajo. You could not pick a Apache and Navajo. You could not pick a more non-representative tribal distribution if more non-representative tribal distribution if you tried, other than one composed entirely you tried, other than one composed entirely of Eskimo.of Eskimo.
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Native American mtDNA Native American mtDNA variationvariation
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mtDNA databasemtDNA database ‘‘Asian’ subset is also not representative Asian’ subset is also not representative
of the geographic origin of Asian of the geographic origin of Asian Americans, based on the 2000 CensusAmericans, based on the 2000 Census
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But I thought they But I thought they validated it?validated it?
For the most part, validation of the For the most part, validation of the various subsets of the mtDNA database various subsets of the mtDNA database involved confirming that the major involved confirming that the major haplogroups of mtDNA variation present haplogroups of mtDNA variation present in the ‘source’ population were also in the ‘source’ population were also present in their American ‘relatives’, present in their American ‘relatives’, sometimes in somewhat similar sometimes in somewhat similar frequenciesfrequencies
Ummmm. What’s a haplogroup?Ummmm. What’s a haplogroup?
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Haplogroups?Haplogroups?
mtDNA lineages can be divided into mtDNA lineages can be divided into major subgroups (haplogroups) based on major subgroups (haplogroups) based on shared ancestry resulting in shared sets shared ancestry resulting in shared sets of mutationsof mutations
Within each haplogroup are individual Within each haplogroup are individual haplotypes of unique combinations of haplotypes of unique combinations of mutations (this is what is utilized in the mutations (this is what is utilized in the inclusion statistics)inclusion statistics)
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Haplogroups?Haplogroups?
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AnalogyAnalogy
Haplogroups are like last namesHaplogroups are like last names Validating the database is basically like asserting that there Validating the database is basically like asserting that there
are ‘Smiths’ in Europe, and there are ‘Smiths’ here too, so are ‘Smiths’ in Europe, and there are ‘Smiths’ here too, so we’ve got a representative samplewe’ve got a representative sample
BUT the FREQUENCY of ‘Smiths’ will vary across the US, as BUT the FREQUENCY of ‘Smiths’ will vary across the US, as well as across Europe, just as the frequencies of haplogroups well as across Europe, just as the frequencies of haplogroups do. So just asserting that there are ‘Smiths’ in both places, do. So just asserting that there are ‘Smiths’ in both places, and even showing that their AVERAGE frequencies are the and even showing that their AVERAGE frequencies are the same across the continents, doesn’t tell you much about how same across the continents, doesn’t tell you much about how representative your sample isrepresentative your sample is
To identify a person more uniquely, you need their To identify a person more uniquely, you need their whole name (their haplotype)whole name (their haplotype) The frequency of Wilmut G. Smith and Jesus A. Smith are The frequency of Wilmut G. Smith and Jesus A. Smith are
going to be significantly different across the US (and across going to be significantly different across the US (and across Europe)Europe)
Because the haplotype is what the inclusion statistic is based Because the haplotype is what the inclusion statistic is based on, this is really the information we needon, this is really the information we need
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But doesn’t the 95% But doesn’t the 95% confidence interval take all confidence interval take all this into account?this into account?
NO!NO! The calculation of a 95% CI ASSUMES The calculation of a 95% CI ASSUMES
that you have a random sample of the that you have a random sample of the population, and that the population is not population, and that the population is not subdividedsubdivided
It is TOTALLY INVALID if your sample is It is TOTALLY INVALID if your sample is not random, and/or the population is not random, and/or the population is subdividedsubdivided
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Han Chinese ExampleHan Chinese Example(Yao et al. 2002)(Yao et al. 2002)
263 unrelated 263 unrelated individuals from individuals from 13 locations 13 locations were typed for were typed for mtDNA mtDNA haplotype, and haplotype, and sorted into sorted into haplogroupshaplogroups
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Han Chinese ExampleHan Chinese Example(Yao et al. 2002)(Yao et al. 2002)
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Han Chinese ExampleHan Chinese Example(Yao et al. 2002)(Yao et al. 2002)
““The comparison of the regional Han mtDNA samples The comparison of the regional Han mtDNA samples revealed an obvious geographic differentiation in the revealed an obvious geographic differentiation in the Han Chinese….Hence, the grouping of different Han Han Chinese….Hence, the grouping of different Han populations into just “Southern Han” and “Northern populations into just “Southern Han” and “Northern Han”…or the use of one or two Han regional Han”…or the use of one or two Han regional populations to stand for all Han Chinese…does not populations to stand for all Han Chinese…does not appropriately reflect the genetic structure of the Han. appropriately reflect the genetic structure of the Han. Intriguingly, despite numerous historically recorded Intriguingly, despite numerous historically recorded migrations and substantial gene flow across Chinese migrations and substantial gene flow across Chinese form the Bronze Age to the present time…differences form the Bronze Age to the present time…differences between geographic regions have been maintained” (p. between geographic regions have been maintained” (p. 649).649).
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mtDNA DatabasemtDNA Database
We simply don’t have the data to examine the significance of We simply don’t have the data to examine the significance of phylogeographic substruction within the US populations at this phylogeographic substruction within the US populations at this pointpoint
However, it is reasonable to assume that the same kind of However, it is reasonable to assume that the same kind of substructure that exists in all studies of other populations that substructure that exists in all studies of other populations that investigate haplotype phylogeography, and many that investigate investigate haplotype phylogeography, and many that investigate haplogroup phylogeography, is also present in the UShaplogroup phylogeography, is also present in the US
Thus, given the small sample size and non-random sampling Thus, given the small sample size and non-random sampling strategy of the mtDNA database, it is unreasonable to assume it strategy of the mtDNA database, it is unreasonable to assume it can provide meaningful estimates of sequence frequencies for the can provide meaningful estimates of sequence frequencies for the calculation of inclusion statistics.calculation of inclusion statistics.
For more info on these issues, see Kaestle, FA, RA Kittles, AL For more info on these issues, see Kaestle, FA, RA Kittles, AL Roth & EJ Ungvarsky (2006) Database Limitations on the Roth & EJ Ungvarsky (2006) Database Limitations on the Evidentiary Value of Forensic Mitochondrial DNA Evidence. Evidentiary Value of Forensic Mitochondrial DNA Evidence. Amer. Amer. Criminal Law Rev.Criminal Law Rev. 43:53-88. 43:53-88.
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Conclusions?Conclusions?
Requirements for preventing and detecting Requirements for preventing and detecting contamination within ancient DNA research are contamination within ancient DNA research are generally more strict than in forensic generally more strict than in forensic applicationsapplications
Even with these requirements, contamination Even with these requirements, contamination ‘slips through’‘slips through’
The federal mtDNA database is currently The federal mtDNA database is currently inadequate for use in inclusion statistics inadequate for use in inclusion statistics calculationscalculations