Construction and evaluation of a novel set of 90 microhaplotypes for forensic applications using NGS technology

Abstract

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.