Mitochondrial point heteroplasmy: insights from deep-sequencing of human replicate samples.

BACKGROUND: Human mitochondrial heteroplasmy is an extensively investigated phenomenon in the context of medical diagnostics, forensic identification and molecular evolution. However, technical limitations of high-throughput sequencing hinder reliable determination of point heteroplasmies (PHPs) with minor allele frequencies (MAFs) within the noise threshold. RESULTS: To investigate the PHP landscape at an MAF threshold down to 0.1%, we sequenced whole mitochondrial genomes at approximately 7.700x coverage, in multiple technical and biological replicates of longitudinal blood and buccal swab samples from 11 human donors (159 libraries in total). The results obtained by two independent sequencing platforms and bioinformatics pipelines indicate distinctive PHP patterns below and above the 1% MAF cut-off. We found a high inter-individual prevalence of low-level PHPs (MAF < 1%) at polymorphic positions of the mitochondrial DNA control region (CR), their tissue preference, and a tissue-specific minor allele linkage. We also established the position-dependent potential of minor allele expansion in PHPs, and short-term PHP instability in a mitotically active tissue. We demonstrate that the increase in sensitivity of PHP detection to minor allele frequencies below 1% within a robust experimental and analytical pipeline, provides new information with potential applicative value. CONCLUSIONS: Our findings reliably show different mutational loads between tissues at sub-1% allele frequencies, which may serve as an informative medical biomarker of time-dependent, tissue-specific mutational burden, or help discriminate forensically relevant tissues in a single person, close maternal relatives or unrelated individuals of similar phylogenetic background.

Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow.

AIM: To evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples. METHODS: Three long-range high-fidelity DNA polymerases (PlatinumTM PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization. RESULTS: PrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization. CONCLUSION: Optimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).

Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines.

Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics.

Expanded Croatian 12 X-STR loci database with an overview of anomalous profiles.

In order to implement X-chromosome short tandem repeat (X-STR) typing into routine forensic practice, reference database of a given population should be established. Therefore we extended already published data with additional 397 blood samples from unrelated Croatian citizens, and analyzed the total of 995 samples (549 male and 446 female) typed by Investigator® Argus X-12 Kit. To test genetic homogeneity of consecutively processed five historic-cultural regions covering the entire national territory, we calculated pairwise Fst genetic distances between regions based on allele and full haplotype frequencies. Since the comparison did not yield any statistically significant difference, we integrated STR profile information from all regions and used the whole data set to calculate forensic parameters. The most informative marker is DXS10135 (polymorphism information content (PIC = 0.929) and the most informative linkage group (LG) is LG1 (PIC = 0.996). We confirmed linkage disequilibrium (LD) for seven marker pairs belonging to LG2, LG3 and LG4. By including LD information, we calculated cumulative power of discrimination that amounted to 0.999999999997 in females and 0.999999005 in males. We also compared Croatia with 13 European populations based on haplotype frequencies and detected no statistically significant Fst values after Bonferroni correction in any LG. Multi-dimensional scaling plot revealed tight grouping of four Croatian regions amongst populations of southern, central and northern Europe, with the exception of northern Croatia. In this study we gave the first extensive overview of aberrant profiles encountered during Investigator® Argus X-12 typing. We found ten profiles consistent with single locus duplication followed by tetranucleotide tract length polymorphism. Locus DXS10079 is by far the most frequently affected one, presumably mutated in eight samples. We also found four profiles consistent with X-chromosome aneuploidy (three profiles with XXX pattern and one profile with XXY pattern). In conclusion, we established integral forensic Croatian X-chromosome database, proved forensic pertinence of Investigator® Argus X-12 Kit for the entire Croatian population and identified locus DXS10079 as a potential duplication hotspot.

Analysis of 12 X-STR loci in the population of south Croatia.

The aim of the study was to assess forensic pertinence of 12 short tandem repeats (STRs) on X-chromosome in south Croatia population. Investigator® Argus X-12 kit was used to co-amplify 12 STR loci belonging to four linkage groups (LGs) on X-chromosome in 99 male and 98 female DNA samples of unrelated donors. PCR products were analyzed by capillary electrophoresis. Population genetic and forensic parameters were calculated by the Arlequin and POPTREE2 software, and an on-line tool available at ChrX-STR.org. Hardy-Weinberg equilibrium was confirmed for all X-STR markers in female samples. Biallelic patterns at DXS10079 locus were detected in four male samples. Polymorphism information content for the most (DXS10135) and the least (DXS8378) informative markers was 0.9212 and 0.6347, respectively. In both male and female samples, combined power of discrimination exceeded 0.999999999. As confirmed by linkage disequilibrium test, significant association of marker pair DXS10074-DXS10079 (P = 0.0004) within LG2 and marker pair DXS10101-DXS10103 (P = 0.0003) within LG3 was found only in male samples. Number of observed haplotypes in our sample pool amounted 3.01, 7.53, 5 and 3.25% of the number of possible haplotypes for LG1, LG2, LG3 and LG4, respectively. According to haplotype diversity value of 0.9981, LG1 was the most informative. In comparison of south Croatia with 26 world populations, pair-wise [Formula: see text] values increase in parallel with geographical distance. Overall statistical assessment confirmed suitability of Investigator® Argus X-12 kit for forensic casework in both identification and familial testing in the population of south Croatia.

Wnt signaling transcription factors TCF-1 and LEF-1 are upregulated in malignant astrocytic brain tumors.

Since the discovery of the TCF/LEF family of transcription factors, their functions have been under intensive investigation in the area of cancer biology. The work presented in this paper focused on the changes in TCF-1 and LEF-1 expression levels in a set of astrocytic brain tumors. Protein expression was detected using immunohistochemistry and then evaluated by Ellipse software (ViDiTo, Slovakia). Statistical evaluations were performed with the SPSS statistical package, version 14.0 (SPSS Inc., Chicago, IL, USA). Strong TCF-1 and LEF-1 expression was observed in 51.6% and 71% of glioblastoma samples. Statistical analysis confirmed significant differences in protein expression levels associated to 3 important values, weak expression of TCF-1, weak expression of LEF-1 and strong expression of LEF-1. Analysis of variances performed on the total sample also indicated significant differences in the values of TCF-1 weak (F=2.804; p=0.045), LEF-1 weak (F=4.255; p=0.008) and LEF-1 strong (F=5.498; p=0.002) with regard to malignancy grade. Thus, glioblastomas were characterized by -in relative terms- the lowest values for weak expression of TCF-1 and LEF-1, combined with the highest values of LEF-1 strong expression. The F-ratios for two variables (LEF-1 strong and LEF-1 weak) indicated that differences between astrocytomas (II, III) and glioblastomas were statistically significant (p<0.02). Discriminant function analysis further showed that strong LEF-1 expression alone could discriminate between astrocytomas (II, III) and glioblastomas. Elevated TCF-1 and LEF-1 expression is characteristic of malignant gliomas. LEF-1, in particular, may serve as a potential marker for malignant transformation.