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Searching the undetected mtDNA variants in forensic MPS data

Posted on 10.10.2020by Mugo

Melchionda F, Stanciu F, Buscemi L, Pesaresi M, Tagliabracci A, Turchi C. Forensic Sci Int Genet. 2020 Nov;49:102399

Abstract:

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.

Keywords: Mitochondrial, DNAMassive, parallel sequencing, Point heteroplasmy, Variants call, Forensic genetics

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Articole Publicații

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

Posted on 30.06.2016by Mugo

Turchi C, Stanciu F, Paselli G, Buscemi L, Parson W, Tagliabracci A. Forensic Sci Int Genet. 2016 Sep;24:136-142. doi: 10.1016/j.fsigen.2016.06.013. Epub 2016 Jun 30. PMID: 27414754.

Abstract:

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.

Keywords: Mitochondrial DNA, Forensic database, Haplogroup, Romanians

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Postere Publicații

mtGenome resolution of the two most common Romanian haplotypes R0 and X2e1b and evaluation of point heteroplasmy detection by massively parallel sequencing

Posted on 20.05.2016by Mugo

Turchi C, Stanciu F, Buscemi L, Tagliabracci A, „mtGenome resolution of the two most common Romanian haplotypes R0 and X2e1b and evaluation of point heteroplasmy detection by massively parallel sequencing”, Poster at Haploid Markers 2016 – Update on DNA variation, 10th International Y Chromosome workshop • 7th International EMPOP meeting. Berlin, May 20-21, 2016.

Abstract:

In the last years has been largely demonstrated that massively parallel sequencing technologies (MPS) offers new possibilities for forensic genetics, both for the more information that may be obtained in a single experiment from unique samples by analyzing combinations of markers, both for the analytical cost-effectiveness. In particular, the full mitochondrial genome (mtGenome) sequencing using MPS has largely applied in the forensic context, as the throughput and sensitivity of the technology resulting in far more efficient data production than can be achieved via Sanger sequencing. Massively parallel sequencing of entire mtGenome increases the power of discrimination, allows more comprehensive heteroplasmy detection, as well as high phylogenetic resolution, with respect to sequence information recovered from the solely control region (CR). Considering this limited range, some samples share identical haplotypes, especially the most frequent mtDNA CR types that are typical of different populations. In an original dataset of 407 Romanian sequences (Turchi C. et al, in press), collected from the general population belonging to four major historical regions Moldavia, Transylvania, Wallachia and Dobruja, we observed 277 (68%) distinct haplotypes of which 220 (79%) were unique. The most common haplotype 16519C, 263G, 315.1C, (haplogroup R0) was shared by twenty-five individuals (6%), while the second most common haplotype 16126C, 16189A, 16223T, 16278T, 16519C, 73G, 195C, 263G, 315.1C (haplogroup X2e1b) was shared by ten individuals (4%). In order to investigate the degree of variation inside these two clades, twenty-one samples were submitted to entire mtGenome sequencing using the Personal Genome Machine (PGM). In the same dataset, point heteroplasmic positions were observed at nine different sites (152Y, 185R, 214R, 225R, 235R, 16093Y, 16172Y, 16189Y and 16399R) in fifteen different samples. The ability to identify and utilize the discrimination potential of heteroplasmy will significantly enhance the value of mtDNA analysis in forensic casework, but at present this condition is commonly disregarded mostly due to the poor sensitivity of the Sanger sequencing in detecting low-level heteroplasmy. Several studies have been carried out recently regarding the threshold for heteroplasmy detection by MPS, being generally suggested that this methodology is more sensitive and accurate. To investigate the level of point heteroplasmy and estimate alleles quantification, fifteen samples showing PHP in the control region were submitted to mtGenome sequencing and the sequences obtained through the PGM are compared to those obtained by Sanger sequencing to determine heteroplasmy detection threshold with both technologies.

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Rubrica de Știință

Calendar Științific 2015

Posted on 17.09.2014by Mugo

Asociația Secular-Umanistă din România (ASUR) este una din asociațiile implicate activ în promovarea științei în România. Am aflat de activitățile desfășurate de ASUR de pe internet, însă ceea ce m-a făcut să apreciez și mai mult eforturile celor implicați a fost strângerea de fonduri și susținerea publicării în România a celebrei cărți scrisă de Carl Sagan – Cosmos și la a cărei lansare am participat în luna mai a.c.

Postere Publicații

mtDNA control region forensic database in the Romanian population and deep investigation of the most frequent haplotypes

Posted on 14.05.2014by Mugo

Turchi C, Stanciu F, Tagliabracci A, „mtDNA control region forensic database in the Romanian population and deep investigation of the most frequent haplotypes”, Poster at DNA in Forensics 2014. 9th Y-User Workshop and 6th EMPOP meeting. Abstract book, Brussels 14-16 mai 2014, P33, p.110.

Abstract:

Romanian population is composed of 88.92% Romanians, 6.5% Hungarians, 3.29% Roma and 1.29% other populations (2011 census). From the historical point of view Romanians are an admixture of local and surrounding populations. Romania can be ided in 4 major historical regions, each with its particular populations influence: Moldavia that during the past was the Eastern Europe border in front of Mongol, Tatar and Ottoman invasions; Transylvania, where the Austro-Hungarian Empire had an important influence; Wallachia, whose population is the result of Roman Empire conquests, the Slav migration from the north, and the Turkish south-east influence; and Dobruja, in the past conquered by Greeks, Romans, Tatars, Turks and Slavs. Previous genetic studies made on Y-STR markers suggest that the Slavic influences were dominant and from the perspective of general population (autosomal markers) the dominant influences were Slavic, Italian, Greek and Turkish; unfortunately there are limited data on mtDNA variation in the general population. In order to analyze the heterogeneity of Romanian population from a mitochondrial lineages point of view and to establish appropriate mtDNA forensic database, we generate a high-quality mtDNA control region data from a Romanian population sample. ~400 healthy Romanian donors, from different regions of the country, were subjected to control region sequence analysis. Two PCR fragments were sequenced by using ten different sequencing primers, according to forensic standards. To ensure high data quality at least a double reading of each site and an independent evaluation of electropherograms were performed. A phylogenetic approach for a posteriori analysis of the mtDNA types was applied and sequences were aligned according to the mitochondrial phylogeny. To increase the utility of mtDNA analysis in forensics, the Personal Genome Machine was used to sequence complete mtGenomes of the most common haplotypes, in order to investigate in more detail specific coding region variations.

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