This document discusses microhaplotypes as a new type of genetic marker for forensic analysis. Microhaplotypes are loci containing 2 or more SNPs within 200 base pairs, making them multi-allelic. This increases their statistical power over individual SNPs. The study aims to validate microhaplotypes for familial searching, mixture analysis, and other forensic applications. It presents materials and methods for evaluating over 50 candidate microhaplotype loci on over 2500 individuals from 54 populations. 31 highly informative loci were selected as a pilot microhaplotype panel.

1. MICROHAPLOTYPES
A POWERFUL NEW TYPE OF
GENETIC MARKER
By:
Mojgan Talebian
1

2. The Study Objectives
2
 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

3. 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

4. SNPs
4
http://www.bioinfoindia.org

5. 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

6. 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

7. 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

8. SNPs and Haplotypes
8

9. 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.

10. Why Microhaplotypes
10
 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

11. 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

12. Microhaplotypes
12

13. The Study Criteria
13
 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

14. Materials and Methods
14
 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. 15

16. 16
Population-specific allele frequencies
Haplotype frequency plots for best 4-SNP microhaps characterized to date

17. Microhaplotype Application
17
 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

18. Mixture Detection
18
 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

19. 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.

20. 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

21. References
21
 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