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.
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
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
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
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/)
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
Hinweis der Redaktion
A coloured SNP in each chromosome forms sets of linked SNP in the respective haplotype