Literature ReviewIntroduction Single nucleotide polymorphisms (SNPs) are changes in the bases of the DNA, occurring at a high density throughout the genome. With millions of validated SNPs and a variety of robust SNP analysis platforms, these markers have been distinguished for their variety of uses. These include ancestry and lineage informative SNPs and phenotype informative SNPs. SNP markers are valuable for analysing challenging forensic sample like those that are very degraded and for augmenting the power of kinship analyses. Kinship analysis is the use of DNA samples to identify potential genetic relations between individuals and has a wide range of applications in forensic science.Novel multi-allelic markers are being promoted to improve current drawbacks of kinship analysis, where valid comparisons cannot be made as a result of factors like limited availability of family members. Continuous research on forensic uses of multi-allelic SNPs is aiming to establish panels to aid in addressing all major forensic DNA questions in one laboratory analysis. This is achievable due to current high-throughput technology, like next generation sequencing.This project will be aimed at investigating the use of non-binary SNPs for complex kinship analysis.Types of Genetic Loci Single nucleotide polymorphisms (SNPs) are changes at single positions in a DNA sequence among individuals and are the most abundant form of human genetic variation (2). Each SNP represents a substitution, insertion or deletion of a nucleotide (DNA building block) and millions of SNP sites have been identified in humans (17). Single nucleotide polymorphisms may be located within coding regions (of genes), non-coding regions or in the intergenic regions (11). Due to their high density, SNPs are deemed valuable markers in the study of human health. These polymorphisms can be used to identify genes associated with complex disease and predict the risk of an individual developing particular diseases, in genome-wide association studies (15).Short tandem repeats (STRs) are the primary DNA markers used in forensic casework, mainly for identification purposes and relationship testing of closely related individuals. SNPs, however, have increasingly become markers of high interest forensically, in cases where additional or alternative genetic marker analyses are required (11)(13). Despite the fact that a larger quantity of binary SNPs are required to achieve the same power of discrimination, the use of SNPs displays advantageous characteristics over the use of STRs. The application of SNPs is favourable in cases where forensic samples contain highly degraded or too little template DNA (8)(13). The success of DNA analysis is highly dependent on the quality of the template DNA. Nevertheless, SNPs can be analysed in shorter sized amplicons than STRs, with a required fragment length of only 60-80bp, allowing for the recovery of information even from damaged samples (20). This is particularly advantageous for samples obtained from human remains of victims of mass disaster. Furthermore, SNPs are more likely to become ‘fixed’ in a population, as they have a much lower mutation rate than STRs, and as a result, tend to be population specific and, therefore, are ancestry informative. This can be beneficial for prediction of an offender’s ethnic origin in criminal investigations (4)(14)(18). In some cases, STRs alone may not be provide sufficient information and the supplementary addition of SNPs will be required to improve the genetic data collected. Moreover, the differences between SNPs and STRs mean that their simultaneous use can provide better insights than if either marker type was to be analysed in isolation (11).Additionally, in addition to the low mutation rate of SNPs, their lack of genetic recombination means they are lineage informative and are very useful for evolutionary studies and kinship analysis (2)(4)(14).Kinship AnalysisKinship analysis is the investigation of genetic relationships between alleged family members and is of crucial value in forensic science. The applications of kinship analysis include paternity testing, settling immigration cases and identifying recovered remains victims of mass disasters, or in missing person enquiries. The standard method used for relationship tests is DNA profiling (using STRs), however in numerous forensic cases more complex relationships have to be considered, where two samples are separated by a number of generations. (6)(9)(13)Successful genetic testing using kinship analysis is limited by a number of factors that affect the evidential weight when determining relatedness. This includes the amount of DNA available for analysis, the availability of close family members for comparison and the available genetic markers. The further the genetic ‘distance’ between individuals, the higher the chances of false negatives/positives or inconclusive results, therefore, the lower the reliability of the relationship test. In order to solve cases involving more distantly related individuals, more information is required and some of the ways this can be achieved include increasing the number of or utilising more informative markers. This increases the power of discrimination resulting in a lower likelihood of two unrelated individuals randomly sharing markers that lead to false positives in kinship analyses. The utilisation of STRs and bi-allelic SNPs can provide enough information for the resolution of first and second-degree relationships. However, the current lineage SNPs have limited power of discrimination and the possibility of false negatives/positives can still be observed. (4)(6)(9)(22)Multi-allelic Loci The vast majority of SNPs are biallelic, however, multi-allelic markers exist where specific sites in the genome consist of more than 2 alleles. In comparison to the large abundance of knowledge and data available on biallelic SNPs, there has been limited success in the study of tri- or tetra-allelic SNPs and they remain insufficiently understood (21). Multi-allelic SNP loci, however, have been established to enable the analysis of mixed and degraded DNA samples (20). Additionally, these sites have been proposed as suitable markers to supplement individual identification, paternity testing and for addressing other forensic matters like family/clan and pedigree/lineage inference (4)(8)(13)(22).The common binary SNPs consist of 2 possible alleles and as a result there are usually 3 possible genotypes. Multi-allelic markers have a higher number of possible alleles. For example, tri-allelic SNPs (consisting of 3 possible alleles) are able to generate 6 possible genotypes, increasing the number of potential genotypes, which increases the power of discrimination. The presence of unusual SNPs, like multi-allelic markers, in a population aids when resolving relationships or estimating the ethnic origin of a sample. The more of these markers used the higher the discriminatory power (4). A higher discrimination power provides a higher resolution in relationship tests, and therefore, the use of these multi-allelic sites is not only plausible, but a promising approach for solving complex cases involving distantly related individuals.Advanced sequencing technologies, like massive parallel sequencing (MPS), otherwise known as next generation sequencing (NGS), have improved the analysis of tri- and tetra-allelic SNPs as well as making the observation of novel multi-allelic SNP loci, such as microhaplotypes, possible (1)(18)(9). Microhaplotypes consist of multiple (two or more) SNPs in close proximity, within 200bp of DNA (6). The Kidd. laboratory first introduced microhaplotypes and promoted its application for identifying pedigree, DNA profiling, and other uses in forensic genetics. The Kidd. laboratory, in addition to other previous research, have been able to compile lists of candidate microhaplotypes, tri- and tetra-allelic SNPs using platforms like the 1000 genomes project (6)(12)(15).The 1000s genome project, launched in 2008, was aimed at founding a public reference database for genetic polymorphisms through multiple approaches applied to a set of diverse individuals from various continental populations. These included whole-genome sequencing, deep targeted-exome sequencing and dense microarray genotyping. While this project is a valuable resource displaying a broad spectrum of DNA polymorphisms, including bi-allelic and multi-allelic SNPs, some of these SNPs are not true tri- or tetra-allelic SNPs. Greatly similar regions may be duplicated in different locations in the genome and, as a result, may generate false SNP predictions. (2)(12)Although multi-allelic SNPs have been promoted as potential forensic markers for DNA analysis, only a limited number have been studied and proven to be suitable for forensic analysis (21). Previous studies have agreed that the use of microhaplotypes and other multi-allelic markers have great prospects for kinship analysis (12)(15)(16)(18)(19). This is because the power of the regular markers used are restricted due to the high mutation rate and stutter of STRs and the low polymorphism of SNPs (12). Microhaplotypes and other multi-allelic loci are emerging forensic genetic markers and more research into them is required, in order to be able to exploit the benefits they provide when used as supplementary markers (22).Next-generation sequencing A promising approach for the identification of multi-allelic sites is through the use of next generation sequencing, also known as, massive parallel sequencing (5). Due to it’s speed, accuracy and read lengths, NGS has been proposed as an alternative to the standard capillary-based methods (6)(18). This technology allows for the rapid analysis of hundreds of markers that are co-amplified and individually sequenced in parallel, with read lengths of up to 400 nucleotides. Sanger sequencing by capillary electrophoresis is the “gold” standard methodology and is highly recognised for DNA sequencing applications, as it provides a high degree of accuracy as well as long-read capabilities. However, a substantial limitation is its inability to produce a phase-known sequence, where the cis/trans relationships can be determined amid the different SNPs. Next-generation sequencing eliminates this obstacle and can determine the allelic combination of multiple SNPs on each chromosome, within a small segment of DNA (3)(6)(9)(18). This allows for the identification of regions, such as microhaplotypes, composed of two to four SNPs that define multi-allelic loci (1)(3).  These microhaplotypes can be used to radically augment the power for relationship inference, and to lower false positive rates, in comparison to the sole use of bi-allelic SNPs (1).This methodology consists of a set of sequencing platforms and Illumina Miseq FGxTM system is noticeably the most preferred by researchers. The fact it contains the most affordable sequencing costs may contribute to this (18)(19).Aim of Study  This principal aim of this project is to assess candidate microhaplotypes, tri- and tetra-allelic SNPs, in order to establish a panel of multi-allelic markers that, when implemented, can improve the resolution of complex relationship testing.The simulation of expected likelihood ratios, attainable for certain relationship comparisons has been used for assessing the influence of the markers for kinship analysis in numerous studies (7)(10)(13)(16). These simulations are carried out using allele frequencies. The distribution of these ratios can be represented on graphs like in figure 1 where the primary end point of this research is shown. Augmenting the power of relationship tests means lowering the false positive rates. The overlap shown in the first LR distribution plot represents an area of uncertainty (false positives/negatives or inconclusive results) and diminishing this area will increase the resolution of kinship analyses.The use of multi-allelic loci serves an important role in analysing challenging forensic samples. The benefits of this research do not only include the ability to distinguish between indirect relatives, but also for family reconstructions for missing persons and the ability to identify remains of disaster victims when the number of family members for comparison is very limited.ReferencesBaetscher, D., Clemento, A., Ng, T., Anderson, E. and Garza, J. (2017). Microhaplotypes provide increased power from short-read DNA sequences for relationship inference. Molecular Ecology Resources, 18(2), pp.296-305.Budowle, B. and van Daal, A. (2008). 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