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Assessment of Illumina® Human mtDNA Genome assay: workflow evaluation with development of analysis and interpretation guidelines
- Source :
- International Journal of Legal Medicine
- Publication Year :
- 2021
- Publisher :
- Springer Science and Business Media LLC, 2021.
-
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.
- Subjects :
- Mitochondrial DNA
Guidelines as Topic
Computational biology
Biology
DNA, Mitochondrial
01 natural sciences
Genome
Workflow
Pathology and Forensic Medicine
03 medical and health sciences
0302 clinical medicine
Humans
030216 legal & forensic medicine
mtDNA control region
Massive parallel sequencing
010401 analytical chemistry
High-Throughput Nucleotide Sequencing
Reproducibility of Results
Sequence Analysis, DNA
Molecular diagnostics
0104 chemical sciences
Minor allele frequency
Genome, Mitochondrial
Quality Score
Human genome
MiSeq
mitochondrial DNA
Nextera XT
evaluation
analysis thresholds
Subjects
Details
- ISSN :
- 14371596 and 09379827
- Volume :
- 135
- Database :
- OpenAIRE
- Journal :
- International Journal of Legal Medicine
- Accession number :
- edsair.doi.dedup.....a790a6fd3a46a811fd20254a30b8e3c6