A 124-plex Microhaplotype Panel Based on Next-generation Sequencing Developed for Forensic Applications

Abstract

Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top A_e values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10^-66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications.

Figure 1

Read counts and percentage of reads representing the alleles for 10 reference samples. Number of effective reads (those called as microhaplotype alleles) are shown in orange, and the total reads are shown in blue.

Figure 2

Average allele coverage ratio (ACR) for each locus. Horizontal black line, number of heterozygotes for each calculated ACR. Error bars represent standard deviations.

Figure 3

Average percentage (%) depth of coverage (DoC) for each locus. Error bars represent standard deviations.

Figure 4

Representative STR profiles and representative microhaplotype genotyping histograms for the mixture experiments. Signal peaks for D16S539-12 and CSF1PO-11 in the 1:6 mixture are indicated as “Allele” and “Stutter”, respectively, for comparison. Numbers under each microhaplotype allele are the numeral allele names assigned to allow the microhaplotype data to be read conveniently.

Figure 5

Histogram of the A_e values for the 124 microhaplotypes.