Testing Accuracy

The target for Hiro Clinic’s testing accuracy (probability of paternity inclusion) is

99.99999999999% or higher is our target.

If we assume a global population of 8 billion people, the probability of a mismatch would be only 0.008 individuals. This result is derived using a statistical measure called the likelihood ratiolikelihood ratio, and it is achievable through the use of STR analysis.

Relationship Between Likelihood Ratio and Probability of Paternity

Because STRs (Short Tandem Repeats) have a wide range of numerical variations, they allow for more precise differentiation.

To achieve this level of accuracy using SNPs (Single Nucleotide Polymorphisms) alone, more than 5,000 SNPs would be required.

Modern Advancements in DNA Paternity Testing Technology

The ABO blood group system was discovered in 1900 and began to be used for paternity exclusion testing in the 1930s. Later, the inclusion of additional blood markers such as the Rh antigen, MNS antigens, and human leukocyte antigens (HLA) improved testing accuracy. However, these methods could not completely eliminate errors.

In 1977, the development of DNA sequencing through the Sanger method enabled direct analysis of DNA, achieving greater statistical accuracy in paternity testing compared to traditional techniques. Today, DNA testing is widely used not only for paternity testing but also in forensic investigations, prenatal testing, and many other applications.

Testing Process

If the test results are intended for use in legal proceedings, the samples from the parties involved are collected at the clinic by a physician. In all other cases, samples can be easily collected at home using a simple and user-friendly kit, and then sent back to the laboratory.

Upon receipt, the samples are registered in the laboratory database. The collected cells then undergo DNA extraction and purification from the cell nuclei. This process is performed using a fully automated, high-throughput system, achieving shorter processing times, lower costs, and higher accuracy compared to traditional DNA testing methods.

Additionally, instead of analyzing the entire genome, the method targets specific marker alleles. This approach maintains high accuracy while significantly reducing testing time and cost.

These specific marker alleles are known as “STRs” (Short Tandem Repeats). After amplification, they are compared to verify biological relationships such as paternity, parentage, or sibling relationships.

What Are STRs?

STRs (Short Tandem Repeats) are short, identical sequence units that are repeated consecutively within a DNA sequence.

Figure 1: In the case of the STR known as CSF1PO, the short sequence “AGAT” is repeated between 5 and 17 times.

Figure 2: A schematic diagram showing three different STRs: D3S1358, D4S2408, and FGA.
There are various types of STRs depending on the specific sequence and location.

STR Analysis Method

Specific polymorphic short tandem repeat (STR) loci are first labeled through a two-step polymerase chain reaction (PCR) process, followed by targeted next-generation sequencing (NGS). The high read depth and sensitivity of this method allow for detection even from samples such as used toothbrushes or drinking straws.

The amelogenin gene is widely used for sex determination in forensic and prenatal diagnostics. In our testing, we enhance the accuracy and reliability of sex determination by analyzing an additional 25 Y-chromosome markers.

We employ the MiSeq FGx Sequencing System, the first next-generation sequencing platform specifically designed, developed, and validated for forensic genomics. After sequencing with this robust genetic panel, an integrated optimized analysis platform—the Universal Analysis Software (UAS)—performs bioinformatics analysis.This platform uses a proprietary alignment algorithm to check not only the STR repeat regions but also the flanking regions adjacent to the primer sequences (both forward and reverse). It identifies loci, alleles, and genotypes based on the entire amplicon sequence, rather than just amplicon size.

Loci with read counts below a specified threshold are excluded, while those above the threshold are considered valid alleles. Furthermore, the sequencing data is rigorously analyzed, taking into account important factors such as allele frequencies in populations, linkage disequilibrium, and the physical proximity of STR loci on the same chromosome.This results in highly sensitive, accurate, and reliable outcomes.

The systems and reagent kits used in this testing have undergone developmental validation and meet the minimum standards set by European guidelines, SWGDAM (Scientific Working Group on DNA Analysis Methods), CODIS (Combined DNA Index System), Interpol, and ESS (European Standard Set).

Details of the bioinformatics analysis performed by the proprietary Universal Analysis Software (UAS) and the statistical algorithms used for kinship analysis are beyond the scope of this article.
If you are interested in the mathematical foundations, please refer to the cited references for an overview of the methods employed.