The application of short and highly polymorphic microhaplotype loci in paternity testing and sibling testing of temperature-dependent degraded samples

Abstract

Paternity testing and sibling testing become more complex and difficult when samples degrade. But the commonly used genetic markers (STR and SNP) cannot completely solve this problem due to some disadvantages. The novel genetic marker microhaplotype proposed by Kidd’s research group combines the advantages of STR and SNP and is expected to become a promising genetic marker for kinship testing in degraded samples. Therefore, in this study, we intended to select an appropriate number of highly polymorphic SNP-based microhaplotype loci, detect them by the next-generation sequencing technology, analyze their ability to detect degraded samples, calculate their forensic parameters based on the collected 96 unrelated individuals, and evaluate their effectiveness in paternity testing and sibling testing by simulating kinship relationship pairs, which were also compared to 15 STR loci. Finally, a short and highly polymorphic microhaplotype panel was developed, containing 36 highly polymorphic SNP-based microhaplotype loci with lengths smaller than 100 bp and Ae greater than 3.00, of which 29 microhaplotype loci could not reject the Hardy-Weinberg equilibrium and linkage equilibrium after the Bonferroni correction. The CPD and CPE of these 29 microhaplotype loci were 1-2.96E-26 and 1-5.45E-09, respectively. No allele dropout was observed in degraded samples incubated with 100°C hot water for 40min and 60min. According to the simulated kinship analysis, the effectiveness at the threshold of 4/−4 reached 98.39% for relationship parent-child vs. unrelated individuals, and the effectiveness at the threshold of 2/−2 for relationship full-sibling vs. unrelated individuals was 93.01%, which was greater than that of 15 STR loci (86.75% for relationship parent-child vs. unrelated individuals and 81.73% for relationship full-sibling vs. unrelated individuals). After combining our 29 microhaplotype loci with other 50 short and highly polymorphic microhaplotype loci, the effectiveness values at the threshold of 2/−2 were 82.42% and 90.89% for relationship half-sibling vs. unrelated individuals and full-sibling vs. half-sibling. The short and highly polymorphic microhaplotype panel we developed may be very useful for paternity testing and full sibling testing in degraded samples, and in combination with short and highly polymorphic microhaplotype loci reported by other researchers, may be helpful to analyze more distant kinship relationships.

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

“…For our 50-plex MH panel, Het values of more than 98% (49/50) of MHs were >0.40, A e values of more than 80% (40/50) MHs > 3.0, and CDP and CPE were 1–3.109 × 10 −49 and 1–8.727 × 10 −16 , respectively ( Supplementary Table S4 ). The results showed that our panel has surpassed the capacity of commonly used 23 STRs [ 50 , 51 ] or 52 SNPs [ 52 ] and several other reported MH panels [ 30 , 53 ], indicating that our panel could be potentially effective for future applications in individual identification, kinship testing, mixture interpretation, and non-invasive prenatal paternity testing (NIPPT) [ 3 , 42 ].…”

A novel approach of kinship determination based on the physical length of genetically shared regions of chromosomes

“…However, its limitations become apparent with those STRs not only in cases involving the determination of relationships with distant degrees (e.g., second-cousins) but also in complex relationships. For example, it is challenging to distinguish full-siblings from half-siblings using autosomal STRs and a standard LR approach (Wen et al 2022 ). However, the ICS-based approach offers a valuable means of determining a relationship based on the calculated ICS value without the need for assumptions about the alleged relationship when evaluating genetic evidence.…”

Bibliometric analysis of kinship analysis from 1960 to 2023: global trends and development

“…The detection of DNA sequence polymorphism has been enhanced, and the exploration of novel genetic markers is steadily advancing. The advantages of MHs in the fields of mixture deconvolution ( Oldoni et al, 2020 ; Tao et al, 2022 ; Yu et al, 2022 ), biogeographic ancestry inference ( de la Puente et al, 2020 ; Tao et al, 2022 ), complex kinship analysis ( Wen et al, 2022 ; Xue et al, 2023 ) and personal identification ( Pu et al, 2017 ) have been extensively investigated. Based on NGS, FGG has played a significant role in identifying unknown remains ( Bertoglio et al, 2020 ), inferring distant relatives ( Kling et al, 2021 ), and solving cold cases ( Phillips, 2018 ).…”

“…These mixed samples pose additional challenges during analysis due to a higher probability of drop-out or drop-in combined with stutter peak ( Gill et al, 2012 ; Bai et al, 2022 ). To overcome these hurdles, MHs have been extensively studied by scholars due to their combined advantages of STR and SNP markers, such as low mutation rate, high polymorphism, short length, and absence of stutter peaks ( Kidd et al, 2013 ; Oldoni et al, 2019 ; Bai et al, 2022 ; Wen et al, 2022 ). Bai et al developed a large panel consisting of 185 MHs to analyze degraded and/or mixed DNA samples demonstrating its utility in conducting parentage, full sibling, and second-degree relative testing, but improvements are necessary to infer more distant relatives (third-degree relatives) ( Bai et al, 2022 ).…”