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

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Abstract

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.

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Data availability

The datasets generated and analysed during this study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors thank all participants in the study for their valuable contributions in the form of samples and detailed informed consents. The authors are also thankful to Oliver Vugrek, PhD, Head of Laboratory for Advanced Genomics, Division of Molecular Medicine at “Ruđer Bošković” Institute, and their laboratory staff for collaboration in concordance study. Theauthors are grateful to Sara Rožić and Ivana Račić, PhD, who made valuable contributions in the experimental part of this study.

Funding

This work was supported by the Ministry of the Interior of the Republic of Croatia.

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Authors and Affiliations

  1. Biology and Fibres Department, Forensic Science Centre “Ivan Vučetić”, Ilica 335, 10000, Zagreb, Croatia

    Viktorija Sukser, Lucija Barbarić & Marina Korolija

  2. Laboratory for Advanced Genomics, Division of Molecular Medicine, “Ruđer Bošković” Institute, Bijenička cesta 54, 10000, Zagreb, Croatia

    Filip Rokić

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  1. Viktorija Sukser
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  2. Filip Rokić
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  3. Lucija Barbarić
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  4. Marina Korolija
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Correspondence to Marina Korolija.

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The authors declare that they have no conflict of interest.

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This study involved samples collected from human participants. All procedures performed in the study were in accordance with the institutional and national ethical standards.

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Sukser, V., Rokić, F., Barbarić, L. et al. Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines. Int J Legal Med 135, 1161–1178 (2021). https://doi.org/10.1007/s00414-021-02508-z

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  • DOI: https://doi.org/10.1007/s00414-021-02508-z

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