Estimate the heterozygote balance of microhaplotype marker with massively parallel sequencing

Abstract

At present, amplifying short tandem repeat (STR) by polymerase chain reaction (PCR) and subsequently genotyping the markers by capillary electrophoresis (CE) have dominated the DNA analysis in crime laboratories. Peak height which is relevant to the amount of starting DNA template is used frequently in both the interpretation of mixture DNA samples and low template DNA samples. However, many factors may affect the results (e.g., the heterozygote balance). When the smaller peak is less than 70% of the height of the larger peak at a locus, it is taken to be an indication that there is more than one contributor of template DNA. As for STR, alleles with a longer sequences are thought to present relatively smaller peak height both because they stutter more but also because they amplify less, which may destroy the heterozygote balance. Microhaplotype, which is genotyped by massively parallel sequencing (MPS), is utilized for their sequence polymorphism, and may perform better in heterozygote balance. Through our screening, we have evaluated 12 loci for their heterozygote balance using average allele coverage ratio (ACR). ACRs were calculated by dividing the lower coverage allele by the higher coverage allele at a locus. 11 of 12 microhaplotype makers had an average ACR of 0.616–0.806.