GENETIC ANALYSIS 21 SHORT TANDEM REPEATS (STR) LOCUS IN MINANGKABAU POPULATION, WEST SUMATERA, INDONESIA

Background: Minangkabau is the majority ethnic group in West Sumatra, Indonesia. West Sumatra is a disaster area, especially earthquakes and the potential for a tsunami. Allele frequency for 21 short tandem repeat locus and genetic variation are not well known. This data is essential for calculating the Paternity Index and Match Probability for forensic identification. Materials and methods: This was an observational study. We analyze the GlobalFiller STR loci in 25 unrelated individuals from Minangkabau ethnic group. The DNA was extracted using a Prefiller kit and amplified with a Global Filler kit by a GeneAmp PCR System, followed by capillary electrophoresis using ABI Prism 3500 Genetic Analyzer. Data analysis was performed by using Easy DNA and FORSTAT software. Results: We observed 162 alleles with allele frequencies between 0.02 – 0.36. The highest expected heterozygosity and the highest power of discrimination were at the SE33 loci, and the highest match probability was at the D2S441 locus. The Chi-square test showed that all STR loci followed Hardy–Weinberg equilibrium (p > 0.05). All loci were highly polymorphic (PIC > 0.5). The combined discrimination capacity of each locus in the population was 99,999%. Conclusion: The 21 STR loci are useful for forensic analysis and population genetic studies of the Minangkabau population.


Introduction
Indonesia, a maritime nation comprising over 17 000 islands straddling the Pacific and Indian Oceans, links mainland Asia with the Pacific world. Relative to its land area, Indonesia is one of the most varied regions regarding ethnic, linguistic, and genetic diversity (Tumonggor et al., 2013).
Minangkabau is the majority ethnic group in West Sumatra, Indonesia. West Sumatra has an area of 42,013 km, and which total population is 5,580,232 people. Earthquakes often strike along Sumatera's west coast, both on land and beneath the sea. The high level of earthquakes, especially those that occur under the sea, has the potential to cause a tsunami. (BPS Sumbar, 2021). . The GlobalFiller STR kit was created by upgrading the 13 CODIS loci to 20 loci and was made available to the laboratories.
Since the last locus of CODIS is located within the ESS (European Standard Set of Loci), the GlobalFillerSTR kit has become a global system combining CODIS and ESS regions. The GlobalFiller STR kit was used in our study because of these distinguishing features.
The first stage in DNA analysis is to determine the DNA profile from the biological samples and the interpretation of data obtained from this DNA. To interpret the evidence obtained from the DNA molecule correctly, it is necessary to know how often genetic signs are seen in the relevant population (Canpolat et al., 2021). The variety and frequency of alleles at each locus can only be determined through studies conducted on each population. So, the allele frequencies of genetic markers used in forensic sciences should be determined for each population, and a database should be established. It is critical to use an accurate society database in the statistical calculations made to evaluate the results of DNA analysis (Goodwin, W., Linacre, A., Hadi, 2007). The study of genetic analysis STR loci of the Minangkabau ethnic group is not widely known.

Materials and methods
Whole blood samples were obtained from 25 unrelated healthy individuals of the Minangkabau population. Informed consent was obtained from all individuals. This research received ethical approval from the research ethics committee of the Faculty of Medicine, Universitas Andalas 414/UN.16.2/KEP-FK/2021.
Genomic DNA was extracted with PreFiller according to a protocol. The DNA quantification process was performed by dripping 1 µl of the DNA sample on the NanoVueTM Plus reading device (GE Healthcare, USA). Making a master mix in a 0.2 ml Eppendorf tube containing 0.5 µl AmpFISTR PCR reaction mix, 0.5 µl AmpFISTR Gold DNA Polymerase, and 5.5 µl AmpFISTR Globalfiller Primer Set is the first step in the amplification method. In a 0.2 ml Eppendorf tube, 15 µl of the master mix was added, followed by 1 µl of DNA and 9 µl of pH 8.0 TE buffer solution until the total volume was 25 µl. Furthermore, for each amplification process, positive and negative controls were used. The negative control contained 15 µl of the master mix and 10 µl of pH 8 buffer TE.
The positive control included 15 µl of the master mix and 10 µl of DNA template (Globalfiller kit). The amplification of each sample, positive and negative controls, was carried out using the GeneAmp PCR System 9700 (Applied Biosystem, USA). The stages of the amplification process carried out were initial heating at 95°C for 11 minutes, followed by 28 cycles, denaturation at 94°C for 1 minute, annealing at 59°C for 1 minute, elongation at 72°C for 1 minute, and final elongation at 60°C for 60 minutes. The amplification of each sample was repeated two times (Thermo Fisher Scientific, 2019).
A total of 1 µl of the amplified sample was placed in a 96-well plate (Applied Biosystems, USA) containing 9.6 µl of HiDi formamide and 0.4 µl of LIZ Size Standard. The well plate was centrifuged for 1 minute at 3000 rpm. Using a thermocycler, GeneAmp PCR System 9700 (Applied Biosystem, USA), samples were denatured at 95 0 C for 2 minutes before being cooled in a freezer at 20 0 C for 3 minutes. Allele readings at the amplified loci were performed by the ABI PRISM 3500 Genetic Analyzer (Applied Biosystem, USA) and analyzed using Genemapper ID-X v1.4 software (Applied Biosystem, USA) (Applied Biosystems, 2010).

Results
The observed allele frequencies for the 21 STR loci in the Minangkabau ethnic group in Indonesia are shown in Table 1. We found 162 alleles with a frequency range 0.02 -0.36. The most frequent allele was 9 in TH01 loci. The most frequent allele types for each locus were: CSF1PO :12, TPOX: 11, TH01: 9, D13S317: 9, D16S539: The variety of alleles is shown in table 2. The most common allele variety is at the SE33 loci, there are 17 types of alleles (13-33.2). The expected heterozygosity (He), power of discrimination, match probability, probability of exclusion, polymorphic information content, and chi-square for Hardy Weinberg equilibrium (HWE) for each locus were calculated in Table 3. The expected heterozygosity ranged from 0.691 (TPOX) to 0.904 (SE33). The power of discrimination ranges from 0.847 for the TPOX locus to 0.983 for the SE33 locus. The probability of exclusion ranges from 0.42 for the TPOX locus to 0.81 for the SE33 locus. Match probability ranges from 0.03 for the TPOX, D5S818, D22S1045 locus to 0.107 for the D2S441 locus. The polymorphic information content ranges from 0,610 (TH01) to 0.690 (D13S317). The combined power of discrimination in the Minangkabau population (CPD) was 92.43 % and the combined match probability (CMP) was 1.089 x 10 -28 . The chi-square test showed that all locus STR loci followed the Hardy Weinberg equilibrium (p > 0,05). All the loci are highly polymorphic (PIC >0,5). The TH01 locus is the fewest polymorphic STR loci (PIC = 0.610), and D13S317 and D19S433 are the most polymorphic (PIC=0.690).   -