Microhaplotype Loci are a Powerful New Type of Forensic Marker
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Microhaplotype Loci are a Powerful New Type of Forensic Marker
K.K. Kidda, A.J. Pakstisa, W.C. Speeda, R. Lagaceb, J. Changb, S. Woottonb, N. Ihuegbub
aDepartment of Genetics, Yale University bHuman Identification Group, Life Technologies
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Disclosure Over many years the Yale group has
collaborated with researchers at Applied Biosystems and many of the supplies used in recent forensics research at Yale have been provided by AB, now part of LifeTech I receive no personal financial benefit from
LifeTech
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09/23/12
Applied Biosystems HID SNP Panel v2.2 (Identity Panel) for PGM
Based on published SNPs with high heterozygosity and low Fst
Genotype match probabilities of 10-31 - 10-35 175 SNPs
35 Y - SNPs
92 - Ken Kidd SNPs
52 - SNPforID SNPlex
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Application: Subject exclusion and investigative leads 1. high discrimination among ancestral groups 2. hair and eye color determination
Based on panels validated on a wide range of population datasets
hair and eye color
SNPs
245 SNPs
Ancestral SNPs
202
45
HID Applications for PGM™ Ancestral and Phenotypic SNP Panel
Partial Overlap
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At the ISFG meeting in Copenhagen in 2007 we defined different types of SNPs needed to
address different forensic questions
IISNPs: SNPs for individual identification AISNPs: SNPs for inference of ancestry PISNPs: SNPs for inference of phenotype LISNPs: haplotypes of SNPs for inference of
lineage/clan/family relationships
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Recent Developments: Proof of Principle
Lineage informative SNPs as multiallelic mini-haplotypes (<10kb in extent) are already being identified and the necessary population data accumulated.
A.J. Pakstis, R. Fang, M.R. Furtado, J.R. Kidd, K.K. Kidd, 2012 Mini-haplotypes as lineage informative SNPs (LISNPs) and ancestry inference SNPs (AISNPs). European Journal of Human Genetics. 20:1148-1154.
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Following developments in sequencing technology we now focus on
microhaplotype loci Microhaplotype loci are multiallelic Composed of 2 or more SNPs in a segment of
DNA less than 200bp in extent, the read length of the new sequencers SNPs show only modest linkage disequilibrium Regions have no recombination hot spot but
possibly historical recombination(s)
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Our microhap studies exist today in two phases
28 unselected microhaplotypes studied on 2612 individuals in our 54 populations. A total of 48 microhaplotypes identified on
the HGDP panel
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Our first phase 28 unselected microhaplotypes studied on
2612 individuals in our 54 populations. All are < 200bp in extent, many < 100bp Typed as individual SNPs by TaqMan and PHASEd All have at least three alleles in almost all pop’s The best are being converted to PGM typing The first 20 are already in ALFRED under the
keyword microhap
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Characterists of the 28 microhaps in 54 populations
The global average heterozygosity = 0.543 21 of the loci have a global average het > 0.5 Random match probabilities range from 2.93 x
10-13 to 1.03 x 10-18
Collectively there is only moderate ancestry information beyond 6 biogeographic regions
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A Microhaplotype studied on ~2600 individuals in 54 population samples
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Some were not good
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An example of a microhap with modest ancestry information
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These 28 unselected microhaplotypes have overall poor ancestry information
7
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Additional Searches Using the empirical pilot data, we were able
to decided on the best criteria to mine larger SNP datasets: 2 or more SNPs within 200bp of each other Low correlation of allele frequencies (to minimize
SNPs in strong linkage disequilibrium) High Fst, either global or regional Maximize global average heterozygosity
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1000 Genomes microhap search Goal: to identify microHaplotypes which had a
high Fst within SE Asia Caveat: use of 1000 Genomes data from
resequencing has a higher genotype error rate than we would prefer to see, so we used the 1000 Genomes data from the Omni25 chip
We pulled out and PHASEd 34 systems and plotted the haplotype frequencies
We had a high success rate for ancestry informative microhaplotypes
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An example of a 124bp microhap
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We found many systems with multiple SNPs within 200bp windows with 4 or more common microhaplotypes present
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HGDP dataset microhap search Dataset which has high genotyping
accuracy Dataset which has greater population
diversity than the 1000 Genomes Caveat: dataset does not have good marker
density (by design)
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While there were fewer multi-SNP microhaplotypes, we still identified several two-SNP microhaplotypes with high informativeness for lineage and ancestry
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The new search criteria are yielding better microhaplotype loci
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Microhaplotype loci can resolve mixtures qualitatively and quantitatively
Because the single read resolves phase and there are large numbers of reads of each chromosome, existence of three or more haplotypes with sufficient reads indicates more than one person in a sample and the relative numbers of reads of each haplotype theoretically indicate the levels of admixture
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Conclusions (by extrapolation) Microhaps are as easy to type by sequencing as
individual SNPs A panel of ̴̴50 selected microhaps can provide
powerful information on familial and clan relationships
Such a panel can serve as individualizing markers with random match probabilities < 10-30
At least 8 biogeographic regions of ancestry can be distinguished easily
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Conclusions (by extrapolation) Microhaplotypes will be the markers of choice for
forensics When marker typing is done by sequencing Provided enough good loci are found Provided there are adequate reference
population data available
These last two efforts will be a major part of our lab’s research over the coming years
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SNP Database Resources SNP allele frequencies (and haplotype frequencies) are
essential for forensic applications of SNP panels ALFRED has over 37 million allele frequency tables We are putting our microhap data into ALFRED to make those
data accessible for the scientific and forensic communities FROG-kb is compiling the forensically relevant frequency data
for the scientific and forensic communities FROG-kb has data for 11 different forensic SNP panels
allowing calculations of likelihoods for individual SNP profiles
ALFRED: the ALlele FREquency Database http://alfred.med.yale.edu
FROG-kb: Forensic Resource/Reference on Genetics - knowledge base http://frog.med.yale.edu
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Acknowledgements Kidd Lab contributors:
Data Collection and Analyses Andrew J. Pakstis
Judith R. Kidd William C. Speed
Eva Straka
Database Design and Curation Haseena Rajeevan
Usha Soundararajan
We thank the many collaborating researchers who helped assemble the samples from diverse populations. Special thanks are due to the many hundreds of individuals who volunteered to give blood samples for studies of gene frequency variation. Our collaborators at Applied Biosystems (LifeTechnologies) have participated in many aspects of these studies
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Acknowledgements
The forensic aspects of this work were funded primarily by NIJ Grants 2010-DN-BX-K225, 2010-DN-BX-K226, and 2007-DN-BX-K197 to KKK awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. Points of view in this presentation are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice.
ALFRED has been funded by NSF grant BCS0938633
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Thank you for your attention
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References Kenneth K. Kidd, Judith R. Kidd, William C. Speed, Rixun Fang, Manohar R. Furtado, Fiona C. Hyland, Andrew J. Pakstis, 2012. Expanding data and resources for forensic use of SNPs in individual identification. Forensic Science International: Genetics. 6:646-652. Andrew J. Pakstis, Rixun Fang, Manohar R. Furtado, Judith R. Kidd, Kenneth K. Kidd, 2012. Mini-haplotypes as lineage informative SNPs (LISNPs) and ancestry inference SNPs (AISNPs). European Journal of Human Genetics. 20:1148-1154. Haseena Rajeevan, Usha Soundararajan, Andrew J. Pakstis, Kenneth K. Kidd, 2012. Introducing the Forensic Research/Reference on Genetics Knowledge Base, FROG-kb. Investigative Genetics 3:18