MICROHAPLOTYPES

A POWERFUL NEW TYPE OF

GENETIC MARKER

By:

Mojgan Talebian

1

The Study Objectives2

To validate the use of sequencing for familial

searching and other forensic questions by SNP-

based, multi-allelic haplotype loci

To validate the feasibility and utility of microhaps

for forensic work

To disentangle mixtures

Single Nucleotide Polymorphism

(SNP)3

A single nucleotide base difference in the DNA sequence

10 million common SNPs are in the human genome, many of which are already annotated in SNP database

Di-allelic and not highly polymorphism

SNPs are so abundant throughout the genome that it is theoretically possible to type hundreds of them

SNP’s sample processing and data analysis may be more fully automated because size-based separation is not required

SNPs4

http://www.bioinfoindia.org

Why SNPs?5

SNPs are the simplest to find, and supplementary to or the

current use of short tandems

the chip technology that allows large numbers of SNPs to be

typed rapidly and cheaply, which is poorly suited for genotyping

STRPs

SNPs are more stable than STRs since unequal

recombination may happen in STRPs loci and new alleles

can be generated

When using capillary electrophoresis for typing STRPs, some

problems such as stutter, allele dropout, low level alleles and dye

artifacts may occur, while these do not occur with SNPs

SNPs offer a much lower mutation rate than STRPs

sets of SNPs or small insertion-deletion polymorphisms (DIPs or

Indels) can provide robust information on biogeographic ancestry

Ion Torrent PGM

Why SNPs cont.6

SNP genotyping is available on very short DNA fragments,

comparable to those that occur in degraded forensic samples

SNPs can enhance the individual identification statistics

panels of SNPs can provide as much individual uniqueness as the

standard CODIS panel of STRPs

Some SNP genotypes are highly correlated with physical phenotypic

traits such as pigmentation of skin, hair, and eye

A disposable

chip

Why Haplotype SNPs?7

Haplotype systems based on multiple SNPs that are closely

linked have been advocated in recent years

Haplotype (haploid + genotype): a set of SNPs on a single

chromosome that are closely linked to be inherited as a unit

SNPs that are molecularly very close will have extremely low

recombination rates

Haplotypes are optimal type of forensically useful DNA

marker, especially family or lineage inference

Haplotyped SNPs allow more efficient inference of family

relationships on a per locus basis because they constitute

multiallelic loci, analogous to the STRPs

SNPs and Haplotypes8

Why Multi-alleles?9

The value of a locus for identifying familial relationships, i.e., lineage information is related to the number of alleles in the relevant population

Multiple alleles make it less likely two unrelated individuals share both alleles by chance ex. Dialleic – two alleles, A and B; only three possible outcomes:

AA, BB, AB

The more heterozygous a locus, the greater the chance that the relevant alleles are uncommon but more likely to be found among close relatives than random or unrelated individuals if the loci are sufficiently heterozygous with multiple alleles,

smaller numbers of loci can be used

These multiallelic loci have great stability, and are as easy to type by sequencing as individual SNPs.

Why Microhaplotypes10

Microhaplotypes are loci with 2 or more SNPs within the limited expanse of a 200 base pair

convert essentially di-allelic markers into multi-allellic systems

Each multi-allelic microhap is clearly more informative than an individual di-allelic SNP, suitable for forensic applications where DNA is degraded

unaffected by recombination

Genotyping microhaps of close SNPs are more accurate and efficient than separately genotyping divided SNPs

Why Microhaplotypes cont.11

Average heterozygosity of microhaplotype is

higher than SNPs

Microhap loci that have at least three alleles,

have high heterozygosity, are globally

informative, with statistical values

By sequencing , rare variants will be seen when

one occurs within a microhap as an unique

allele, missed when the SNPs are typed

individually

Microhaplotypes12

The Study Criteria13

Use of sequencing for forensics; by identifying a large number of SNP-based, multi-allelic haplotype loci

A minimum criterion for a microhaplotype locus is at least three haplotypes (alleles) within a region smaller than 200 bp

Those microhaps with the higher global average heterozygosity than any of the SNPs alone have been included in this study

Mutation rate of these microhaps are less than the mutation rate of the forensic STRs

Materials and Methods14

Microhaplotypes evaluated on >2500 individuals in 54 populations

Out of over 50 candidate loci evaluated on these 54 populations, 31 loci selected as the pilot microhap panel

The size (molecular extent) range of the 31 microhaps is 18 bp to 201 bp with an average of 107.5 bp

Each individual SNP typed by TaqMan and phased to predict haplotypes

The allele (haplotype) frequencies entered in ALFRED, allele frequency database, under the keyword microhap (https://alfred.med.yale.edu/)

15

16

Population-specific allele frequencies

Haplotype frequency plots for best 4-SNP microhaps characterized to date

Microhaplotype Application17

For routine applications, a microhap panel

must consist of sufficient appropriate loci

The selection of microhaps, depend on its

applications

Kinship/lineage inference

Ancestry inference, population grouping

Individual identification

Mixture detection

Level of heterozygosity, Fst value and Ae value

Mixture Detection18

microhaplotypes can detect and deconvolute mixtures in a

sample as they are multi-allelic with the potential to quantify

the components

With many loci multiplexed and with more loci consisting of three

SNPs defining four or more haplotypes, the microhaps become

powerful markers to identify and quantify components of mixtures

With MPS, even very low levels of a second contributor to a

mixture could result in multiple reads with a unique

combination of SNP alleles (a unique haplotype) on single

reads, giving multiple confirmation of the presence of more

than one DNA.

In contrast to many evaluations of mixtures using STRPs that

require decisions surrounding stutter peaks and low RFU peaks

in mixtures, MPS microhaps are clearly superior

Mixture Detection cont.19

As more heterozygous loci with more alleles are used, the

probability of identifying a mixture rapidly approaches

certainty assuming the sensitivity is great enough

With allele (haplotype) frequencies defined in multiple

populations, computer software should be able to accurately

predict the likelihood and levels of mixture based on observing

more than two sequence types at a locus and the numbers of

occurrences of each type.

Conclusion 20

Microhap panels have the potential to become

the major forensic tools because of their

statistical power

The availability of low-cost methods for

detecting SNPs and sequencing will make

microhap panels an increasingly attractive

alternative to STRs in forensic applications

Further development of microhaps are clearly

needed

References21

K.K. Kidd, A.J. Pakstis, W.C. Speed, R. Lagace, J. Chang, S. Wootton, N.Ihuegbu, 2013. Microhaplotype loci are a powerful new type of forensic marker.Forensic Science International: Genetics Supplement Series 4:e123-e124

Kidd, Kenneth K. et al., 2014. Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics. Forensic Science International: Genetics, Volume 12 , 215 - 224

Masaki Hashiyada, 2011. DNA biometrics, Biometrics, Dr. Jucheng Yang (Ed.), InTech, DOI: 10.5772/18139. Available from: http://www.intechopen.com/books/biometrics/dna-biometrics

Kidd, K. K., 2016. Proposed nomenclature for microhaplotypes. Human Genomics, 10, 16. http://doi.org/10.1186/s40246-016-0078-y

Katherine Butler et al., 2015. Performance of a next generation sequencing SNP assay on degraded DNA Gettings. Forensic Science International: Genetics, Volume 19 , 1 – 9. http://dx.doi.org/10.1016/j.fsigen.2015.04.010