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

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

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

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

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

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.

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)

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

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

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

A Microhaplotype studied on ~2600 individuals in 54 population samples

Some were not good

An example of a microhap with modest ancestry information

These 28 unselected microhaplotypes have overall poor ancestry information

7

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

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

An example of a 124bp microhap

We found many systems with multiple SNPs within 200bp windows with 4 or more common microhaplotypes present

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)

While there were fewer multi-SNP microhaplotypes, we still identified several two-SNP microhaplotypes with high informativeness for lineage and ancestry

The new search criteria are yielding better microhaplotype loci

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

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

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

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

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

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

Thank you for your attention

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