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

Autori
Sukser, Viktorija ; Rokić, Filip ; Barbarić, Lucija ; Korolija, Marina

Izvornik
International journal of legal medicine (0937-9827) 135 (2021); 1161-1178

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
MiSeq ; mitochondrial DNA ; Nextera XT ; evaluation ; analysis thresholds

Sažetak
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 assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx TM 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 TM produces repeatable and reproducible results, independent of 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 sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, primarily in forensic genetics and molecular diagnostics.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA