Searching the undetected mtDNA variants in forensic MPS data.

The efficiency of MPS in forensic mtDNA analysis has been thoroughly proven, although a reliable and well established data evaluation still remains a critical point. Numerous bioinformatics tools have been developed, but most of them require specific operating systems and high costs, while free open-source programs with user-friendly interfaces are few. In this study, 43 full mtGenomes were sequenced using the Ion Personal Genome Machine™ (PGM™) System and analyzed utilizing the plug-in Variant Caller (TVC) of the Ion Torrent Software Suite and the mtDNA-Server (mDS), a free web-based mitochondrial analysis tool for MPS data. The outcomes of these two different analysis tools were compared to variants noted after manual inspection of the aligned reads performed using Integrative Genomics Viewer (IGV). The comparison highlighted the presence of thirty-nine discordant variant calls, which were resolved by Sanger sequencing that confirmed the presence of all variants, except for 7 deletions. The combined adoption of IGV and Sanger type sequencing confirmatory steps, in addition of TVC and mDS analysis, resulted in a more accurate variants assignment with the detection of 32 additional true polymorphisms, which were noted in the final dataset. Regarding the heteroplasmy issue, out of a total of thirty heteroplasmic variants, twenty-eight were detected by the TVC, while the mDS detected twenty-two. Overall, none of the used bioinformatics tools were the perfect choice and a secondary analysis with an expert's opinion in complete mtGenome MPS data evaluation is still required in forensic genetic analysis.

The mitochondrial DNA makeup of Romanians: A forensic mtDNA control region database and phylogenetic characterization.

To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population.

Genetic parameters and allele frequencies of five new European Standard Set STR loci (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in the population of Romania.

AIM: To establish allele frequencies and genetic parameters for 5 new European Standard Set short tandem repeat (STR) loci in the population of Romania and to compare them with those in other populations. METHODS: DNA was isolated using QIAamp 96 DNA Swab BioRobot Kit and Chelex 100 methods. Polymerase chain reaction amplification was done using Investigator ESSplexPlus Kit (D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, and vWA). For DNA typing, Applied Biosystems 3500/3500xL Genetic Analyzer was used. Statistical analysis was done using Powerstats, GDA, and Arlequin software. RESULTS: Power of discrimination and polymorphism information content was highest for two new ESS loci, D1S1656 and D12S391. Comparison of allele frequencies for 5 new ESS loci in Romanian population with previously published population data showed significant differences for all compared populations, with the exception of Hungary. Geographically more distant populations, such as Spain, Sweden, United Kingdom, Germany, and Portugal differed more than closer populations. CONCLUSION: New ESS STR loci are very useful for the analysis of forensic samples (persons or traces) due to their characteristics (shortness and high polymorphism). In comparisons with other common STR markers, they have a higher power of discrimination and also higher polymorphism information content, and could be used in any national DNA database.

Population data for Y-chromosome haplotypes defined by 17 STRs in South-East Romania.

Haplotypes and allele frequencies for 17 STR loci included in AmpFlSTR YFiler kit (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATAH4, DYS437, DYS438 and DYS448) were determined in a sample of 122 unrelated males living in the South-East Romania. Genetic distances had been calculated and dendrograms had been generated for South-East Romanian population and other eighteen surrounding populations.

Allele frequencies of 15 STR loci in Moldavia region (NE Romania).

Allele frequencies for 15 STR loci included in AmpFlSTR Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) were determined in a sample of 1321 unrelated individuals living in the region of Moldavia (NE Romania). No deviations from Hardy-Weinberg equilibrium were observed (only after applying the Bonferroni correction in the cases of D2S1338). Genetic parameters of forensic interest were calculated and comparison with geographically nearby populations was performed.

Population data for 15 short tandem repeat loci from Wallachia Region, South Romania.

AIM: To determine allele frequencies' distribution for 15 short tandem repeat (STR) loci in a population sample of 1910 unrelated individuals from the region of Wallachia, South Romania. METHODS: DNA was isolated using Chelex 100 method and an adapted version of AGOWA mag DNA Isolation Kit Sputum. Polymerase chain reaction amplification was done using AmpFlSTR Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). For DNA typing, ABI PRISM 3100 Genetic Analyzer was used. Genetic parameters of forensic interest were calculated and comparisons with geographically close populations were performed. RESULTS: With the exception of vWA locus (P=0.001), no other significant deviations from Hardy-Weinberg expectations were found. Single locus comparisons with data on geographically close populations showed significant differences between the population of Wallachia and the population of Bucharest area, Greece, Turkey, Italy, Hungary, Belarus, and Poland, but no differences were found from the population from Croatia and Serbia. CONCLUSION: According to 15 analyzed STR loci, the population of Wallachia region was found to be genetically more similar to Slavic populations of Croatia and Serbia than to other surrounding populations.

STR data for the AmpFlSTR Identifiler from Dobruja region (SE Romania).

Allele frequencies for 15 STR loci included in AmpFlSTR Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) were determined in a sample of 569 unrelated individuals living in the region of Dobruja (SE Romania). No deviations from Hardy-Weinberg equilibrium were observed. Genetic parameters of forensic interest were calculated and comparison with geographically nearby populations was performed.

Comprehensive STR data for the AmpFlSTR Identifiler from Transylvania (NW Romania).

Allele frequencies for 15 STR loci included in AmpFlSTR Identifiler kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) were determined in a sample of 1977 unrelated individuals living in the region of Transylvania (NW Romania). No deviations from Hardy-Weinberg equilibrium were observed with the exception of D3S1358 and D16S539. Genetic parameters of forensic interest were calculated and comparison with geographically nearby populations was performed.