Microhaplotypes in forensic genetics

doi:10.1016/j.fsigen.2018.09.009

Forensic Science International: Genetics

Volume 38, January 2019, Pages 54-69

https://doi.org/10.1016/j.fsigen.2018.09.009 Get rights and content

Highlights

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Historical background of microhaplotypes (microhaps, MHs).
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Features of MH markers, methods and technologies used for their analysis.
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Current applications of MHs for human identification, mixture deconvolution, and ancestry inference.
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Perspectives of microhaps for missing person identification, relationship testing, medical/and non-human DNA analysis.

Abstract

Microhaplotype loci (microhaps, MHs) are a novel type of molecular marker of less than 300 nucleotides, defined by two or more closely linked SNPs associated in multiple allelic combinations. The value of these markers is enhanced by massively parallel sequencing (MPS), which allows the sequencing of both parental haplotypes at each of the many multiplexed loci. This review describes the features of these multi-SNP markers and documents their value in forensic genetics, focusing on individualization, biogeographic ancestry inference, and mixture deconvolution. Foreseeable applications also include missing person identification, relationship testing, and medical diagnostic applications. The technique is not restricted to humans.

Section snippets

Discovery of haplotype blocks in the human genome

The term ‘haplotype’ was first introduced by Ruggero Ceppellini in the late 60 s to describe alleles within the human leucocyte antigen (HLA) region that are inherited together as a block [1]. Twenty years later the human genome project (HGP) launched an unprecedented international collaboration [2] foundational to the study of human genetics and biomedical research. The early work focused on mapping of human and mouse genes and sequencing the genomes of significantly smaller and easily studied

Microhaplotypes: a new type of genetic marker

Previous investigations have illustrated the utility of multiple closely linked SNP-based markers in anthropology for population relationship and their capacity to provide a plausible explanation for the pattern of recent human variation [39,[74], [75], [76], [77], [78]]. In addition, multi-allelic SNPs have been promoted as suitable markers for addressing relevant forensic questions such as family/clan, lineage inference, and individual identification [30,79,80]. Aiming to complement current

Applications of microhaplotypes in forensic genetics

Microhaplotypes are a new promising and versatile tool that can be used to simultaneously extract many types of relevant forensic information from one or more individuals contributing to a DNA sample. The multi-faceted nature of microhaps makes them an appealing forensic marker for both DNA profiling and investigative genetics. Indeed, the advent of MPS has enhanced the value of microhaps beyond the original aim of identifying lineage-clan-family relationships by widening the spectrum of

Concluding remarks

The very rapid transition from minihaplotype to microhaplotype markers is analogous to the early forensic shift from minisatellite to microsatellite DNA polymorphisms in the late nineties. Microhaps are a powerful and comprehensive molecular tool that provides a great wealth of information relevant to addressing different forensic and human genetic diversity questions. At least for the foreseeable future, microhaps have the potential to enhance ancestry prediction and mixture deconvolution

Disclosure statement

The authors declare no conflict of interest.

Acknowledgements

The authors would like to acknowledge the three anonymous reviewers for their useful comments on this manuscript. This work was supported in part by the National Institute of Justice through grant No. 2017-DN-BX-0164 awarded to Daniele Podini and grant No. 2015-DN-BX-K023 to Kenneth K. Kidd and the Swiss National Science Foundation through grant No. 2017-P2LAP3_174742 awarded to Fabio Oldoni.

References (139)

L.L. Cavalli-Sforza et al.
Call for a worldwide survey of human genetic diversity: a vanishing opportunity for the Human Genome Project
Genomics
(1991)
K.M. Weiss et al.
Linkage disequilibrium and the mapping of complex human traits
Trends Genet.
(2002)
M.P. Donnelly et al.
The distribution and most recent common ancestor of the 17q21 inversion in humans
Am. J. Hum. Genet.
(2010)
J. Yamtich et al.
Population-specific variation in haplotype composition and heterozygosity at the POLB locus
DNA Repair (Amst.)
(2009)
C. Phillips et al.
SNPforID Consortium, inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs
Forensic Sci. Int. Genet.
(2007)
K.K. Kidd et al.
Developing a SNP panel for forensic identification of individuals
Forensic Sci. Int.
(2006)
K.K. Kidd et al.
Expanding data and resources for forensic use of SNPs in individual identification
Forensic Sci. Int. Genet.
(2012)
B.F.B. Algee-Hewitt et al.
Individual identifiability predicts population identifiability in forensic microsatellite markers
Curr. Biol.
(2016)
C. Phillips
Forensic genetic analysis of bio-geographical ancestry
Forensic Sci. Int. Genet.
(2015)
O. Bulbul et al.
Improving ancestry distinctions among Southwest Asian populations
Forensic Sci. Int. Genet.
(2018)

M. Fondevila et al.

Revision of the SNPforID 34-plex forensic ancestry test: assay enhancements, standard reference sample genotypes and extended population studies

Forensic Sci. Int. Genet.

(2013)

K.B. Gettings et al.

A 50-SNP assay for biogeographic ancestry and phenotype prediction in the U.S. population

Forensic Sci. Int. Genet.

(2014)

O. Bulbul et al.

Evaluating a subset of ancestry informative SNPs for discriminating among Southwest Asian and circum-Mediterranean populations

Forensic Sci. Int. Genet.

(2016)

C.-X. Li et al.

A panel of 74 AISNPs: improved ancestry inference within Eastern Asia

Forensic Sci. Int. Genet.

(2016)

K.K. Kidd et al.

Progress toward an efficient panel of SNPs for ancestry inference

Forensic Sci. Int. Genet.

(2014)

C. Phillips et al.

Building a forensic ancestry panel from the ground up: the EUROFORGEN Global AIM-SNP set

Forensic Sci. Int. Genet.

(2014)

C. Phillips et al.

Eurasiaplex: a forensic SNP assay for differentiating European and South Asian ancestries

Forensic Sci. Int. Genet.

(2013)

M. Kayser

Forensic DNA Phenotyping: predicting human appearance from crime scene material for investigative purposes

Forensic Sci. Int. Genet.

(2015)

M. Kayser et al.

DNA-based prediction of human externally visible characteristics in forensics: motivations, scientific challenges, and ethical considerations

Forensic Sci. Int. Genet.

(2009)

L. Chaitanya et al.

The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation

Forensic Sci. Int. Genet.

(2018)

S. Walsh et al.

Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage

Forensic Sci. Int. Genet.

(2014)

S. Walsh et al.

Developmental validation of the IrisPlex system: determination of blue and brown iris colour for forensic intelligence

Forensic Sci. Int. Genet.

(2011)

Y. Ruiz et al.

Further development of forensic eye color predictive tests

Forensic Sci. Int. Genet.

(2013)

S. Walsh et al.

The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA

Forensic Sci. Int. Genet.

(2013)

A.A. Westen et al.

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

Forensic Sci. Int. Genet.

(2009)

C. Phillips et al.

Tetra-allelic SNPs: informative forensic markers compiled from public whole-genome sequence data

Forensic Sci. Int. Genet.

(2015)

S.A. Tishkoff et al.

A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations

Am. J. Hum. Genet.

(1998)

J.M. Butler et al.

Report on ISFG SNP Panel Discussion

Forensic Sci. Int. Genet. Suppl. Ser. (Progress in Forensic Genetics 12)

(2008)

K.K. Kidd et al.

Microhaplotype loci are a powerful new type of forensic marker

Forensic Sci. Int. Genet. Suppl. Ser.

(2013)

K.K. Kidd et al.

Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics

Forensic Sci. Int. Genet.

(2014)

K.K. Kidd et al.

Evaluating 130 microhaplotypes across a global set of 83 populations

Forensic Sci. Int. Genet.

(2017)

C. Børsting et al.

Next generation sequencing and its applications in forensic genetics

Forensic Sci. Int. Genet.

(2015)

K.K. Kidd et al.

Genetic markers for massively parallel sequencing in forensics

Forensic Sci. Int. Genet. Suppl. Ser.

(2015)

M. Stephens et al.

A new statistical method for haplotype reconstruction from population data

Am. J. Hum. Genet.

(2001)

M. Stephens et al.

A comparison of bayesian methods for haplotype reconstruction from population genotype data

Am. J. Hum. Genet.

(2003)

J. Marchini et al.

International HapMap Consortium, A comparison of phasing algorithms for trios and unrelated individuals

Am. J. Hum. Genet.

(2006)

C. Turchi et al.

A microhaplotypes panel for forensic genetics using massive parallel sequencing

Forensic Sci. Int. Genet. Suppl. Ser.

(2017)

J. Zhu et al.

Genotyping microhaplotype markers through massively parallel sequencing

Forensic Sci. Int. Genet. Suppl. Ser.

(2017)

H. Wang et al.

NGS technology makes microhaplotype a potential forensic marker

Forensic Sci. Int. Genet. Suppl. Ser.

(2015)

Y. Pu et al.

Microhaplotype: ability of personal identification and being ancestry informative marker

Forensic Sci. Int. Genet. Suppl. Ser.

(2017)

R. Ceppelini et al.

F.S. Collins

The human genome project: lessons from large-scale biology

Science (80-.)

(2003)

F.R. Blattner et al.

The complete genome sequence of Escherichia coli K-12

Science

(1997)

C. elegans sequencing consortium, genome sequence of the nematode C. elegans: a platform for investigating biology

Science

(1998)

M.D. Adams et al.

The genome sequence of Drosophila melanogaster

Science

(2000)

R.D. Fleischmann et al.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd

Science

(1995)

A comprehensive genetic linkage map of the human genome. NIH/CEPH Collaborative Mapping Group

Science

(1992)

J.D. McPherson et al.

International Human Genome Mapping Consortium, a physical map of the human genome

Nature

(2001)

W.F. Dietrich et al.

A comprehensive genetic map of the mouse genome

Nature

(1996)

J.C. Venter et al.

The sequence of the human genome

Science (80-.)

(2001)

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Massively parallel sequencing (MPS) techniques were developed approximately 15 years ago. Meanwhile, several MPS kits for forensic identification, phenotypic information, ancestry, and mitochondrial DNA analysis have been developed and their use has been established. Sequencing short tandem repeats (STRs) has certain advantages over the currently used length-based genotyping methods, which are based on PCR amplification followed by capillary electrophoresis (CE). MPS is more discriminative and includes the possibility of testing high numbers of targets (> 100), different types of markers [STRs and single nucleotide polymorphisms (SNPs)], as well as the use of smaller amplicons (< 300 bp). This study evaluated in 24 experimental runs the Precision ID GlobalFiler™ NGS STR panel v2 from ThermoFisher, which targets 31 autosomal STRs, amelogenin, and three Y-markers (one STR, SRY, and Yindel). Single-source samples were used in 18 experimental runs, for systematic evaluation. These included assessing library preparation benchmark conditions, limited DNA input, as well as testing repeatability, number of samples per run, and degraded DNA samples. Full profiles were consistently obtained from as little as 50 pg DNA input. Using the optional recovery PCR method improved outcomes for samples with low DNA input. Full profiles were also obtained from severely degraded DNA samples with degradation indices (DI) of > 60. In addition, six experimental runs were performed testing various two-person mixtures with mixture ratios ranging from 1:20 to 20:1. Major and minor contributors were distinguishable by their read counts (coverage), because less DNA input yielded lower read counts, analogous to the traditional CE technology, where less DNA produces lower peak heights. Mixture ratios of approximately 1:1 were indistinguishable, while a greater imbalance, i.e., higher mixture ratios, made the mixture more distinguishable between major and minor contributors. Based on this information, the highest success rate of correctly deconvoluted four-allelic loci was from mixtures with 1:3 ratios. At higher mixture ratios, the drop-out rate of the minor contributor increased, reducing the number of four-allelic loci.
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Biological traces discovered at crime scenes hold significant significance in forensic investigations. In cases involving mixed body fluid stains, the evidentiary value of DNA profiles depends on the type of body fluid from which the DNA was obtained. Recently, coding region polymorphism analysis has proved to be a promising method for directly linking specific body fluids to their respective DNA contributors in mixtures, which may help to avoid "association fallacy" between separate DNA and RNA evidence. In this study, we present an update on previously reported coding region Single Nucleotide Polymorphisms (cSNPs) by exploring the potential application of coding region Insertion/Deletion polymorphisms (cInDels). Nine promising cInDels, selected from 70 mRNA markers based on stringent screening criteria, were integrated into an existing mRNA profiling assay. Subsequently, the body fluid specificity of our cInDel assay and the genotyping consistency between complementary DNA (cDNA) and genomic DNA (gDNA) were examined. Our study demonstrates that cInDels can function as important multifunctional genetic markers, as they provide not only the ability to confirm the presence of forensically relevant body fluids, but also the ability to associate/dissociate specific body fluids with particular donors.
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Microhaplotypes (MHs), small sets of linked single nucleotide polymorphisms (SNPs), are becoming a valuable tool for paternity testing, personal identification and other different forensic purposes due to their advantages of both short tandem repeats (STRs) and SNPs. However, only a small part of MHs with small segments have been developed and reported so far. And the current population genetic data of MHs are still insufficient. MHs with small segments possess unique advantages in mixture deconvolution, degradation material identification, noninvasive prenatal paternity testing and even medical tumor diagnostic applications. In the present study, a set of 90 autosomal MHs whose PCR amplicon lengths are from 90–150 bp, of which 58 MHs are less than or equal to 100 bp are selected, and assembled into an amplification multiplex system optimized for Ion S5™ System for forensic application. Genetic diversity study of 90 MHs in the populations from different intercontinental regions shows that the polymorphism information content (PIC) values of 83 MHs are greater than 0.4 in populations from East Asia (EAS), and the average PIC value of 90 MHs is greater than 0.5. A total of EAS populations shows the highest cumulative match probability (CMP) and cumulative probability of exclusion (CPE) values in five intercontinental populations. The CMP and CPE values of 90 MHs in EAS are 1.1688 × 10⁻⁵⁴ and 0.999999999998954. The informativeness for assignment (In) values of the 90 MHs are calculated based on data from five intercontinental populations, and the In values of 20 MHs have greater than 0.1, indicating that the 20 MHs are high effectiveness in distinguishing different intercontinental populations, which can be used as candidate ancestry informative markers. Further, we have studied the polymorphisms of the 90 MHs based on 224 unrelated individuals of Henan Han population, China, and obtained the frequency data of the 90 MHs. In the Henan Han population, the effective number of alleles (Ae) of the 90 MHs ranges from 1.7649 (MH45) to 3.9792 (MH50), and the Ae values of 10 MHs reach to 3.0; the Ae values of 80 MHs are greater than 2, and the average Ae value for these MHs is 2.422. The average expected heterozygosity, observed heterozygosity, PIC, matching probability, discrimination power and probability of exclusion values of 90 MHs in the Henan Han population are 0.5788, 0.5851, 0.5039, 0.2608, 0.7392 and 0.2806, respectively. The CMP value of 90 MHs in the study population is less than 10⁻⁵⁴, and their CPE value reaches 0.999999999999999923. Moreover, the results of the depth of coverage, allele coverage ratio and noise level indicate that the 90 MH amplification system has well sequencing performance, and the sequencing results are reliable. The results indicate the 90 MHs show higher polymorphisms in the study population. The present panel can be well used in paternity testing and individual identification in the study population and even the populations from EAS.
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Microhaplotype (MH), as an emerging type of forensic genetic marker in recent years, has the potential to support multiple forensic applications, especially for mixture deconvolution and biogeographic ancestry inference. Herein, we investigated the genotype data of 74 MHs included in a novel MH panel, the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel, in three Chinese Sino-Tibetan populations (Han, Tibetan, and Yi) using the Ion Torrent semiconductor sequencing. The sequencing performance, allele frequencies, effective number of alleles (A_e), informativeness (I_n), and forensic parameters were subsequently estimated and calculated. In addition, principal component analysis (PCA) and structure analysis were performed to explore the population relationships among the three populations and the ancestry component distribution. Overall, this novel MH panel is robust and reliable, and has an excellent sequencing performance. The A_e values ranged from 1.0126 to 7.0855 across all samples, and 75.68 % of MHs had A_e values >2.0000. Allele frequencies at some loci varied considerably among the three studied populations, and the mean I_n value was 0.0195. Moreover, the genetic affinity between Tibetans and Yis was closer than that between Tibetans and Hans. The aforementioned results suggest that the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel is highly polymorphic in three investigated populations and could be used as an effective tool for human forensics. Although these 74 MHs have demonstrated the competency in continental population stratification, a higher resolution for distinguishing intracontinental subpopulations and a more comprehensive database with sufficient reference population data still remain to be accomplished.

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Microhaplotypes in forensic genetics

Highlights

Abstract

Section snippets

Discovery of haplotype blocks in the human genome

Microhaplotypes: a new type of genetic marker

Applications of microhaplotypes in forensic genetics

Concluding remarks

Disclosure statement

Acknowledgements

Genomics

Trends Genet.

Am. J. Hum. Genet.

DNA Repair (Amst.)

Forensic Sci. Int. Genet.

Forensic Sci. Int.

Forensic Sci. Int. Genet.

Curr. Biol.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Am. J. Hum. Genet.

Forensic Sci. Int. Genet. Suppl. Ser. (Progress in Forensic Genetics 12)

Forensic Sci. Int. Genet. Suppl. Ser.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet.

Forensic Sci. Int. Genet. Suppl. Ser.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Am. J. Hum. Genet.

Forensic Sci. Int. Genet. Suppl. Ser.

Forensic Sci. Int. Genet. Suppl. Ser.

Forensic Sci. Int. Genet. Suppl. Ser.

Forensic Sci. Int. Genet. Suppl. Ser.

The human genome project: lessons from large-scale biology

Science (80-.)

The complete genome sequence of Escherichia coli K-12

Science

C. elegans sequencing consortium, genome sequence of the nematode C. elegans: a platform for investigating biology

Science

The genome sequence of Drosophila melanogaster

Science

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd

Science

A comprehensive genetic linkage map of the human genome. NIH/CEPH Collaborative Mapping Group

Science

International Human Genome Mapping Consortium, a physical map of the human genome

Nature

A comprehensive genetic map of the mouse genome

Nature

The sequence of the human genome

Science (80-.)