Capability of the iSeq 100 sequencing system from Illumina to detect low-level substitutions in the human mitochondrial genome

The significance of mtDNA heteroplasmy in forensic and medical genetics has increased recently because massively parallel sequencing (MPS) technologies enable more accurate and precise detection of minority nucleotide variants. Recent reports have shown that detection of low-level substitutions may depend on library preparation or sequencing protocol, and can vary for different MPS platforms. The MiSeq (Illumina) and Ion S5 (Thermo Fisher Scientific) are mainly used for heteroplasmy detection, but no data are available regarding the iSeq 100, an Illumina platform of the smallest throughput. Notably, unlike the other systems, the machine utilizes sequencing by synthesis one-channel chemistry to determine DNA sequences. Thus, it is important to verify the capability of the iSeq 100 system to determine mitochondrial haplotypes and detect heteroplasmic substitutions. In this study, previously determined entire mitochondrial genomes were sequenced with the iSeq 100 system. Each mitogenome was sequenced twice, giving approximately 2000x and 10,000x coverage. All homoplasmic mutations and minority variants above the 19 % level detected with the iSeq 100 system were also observed after dideoxy sequencing. Moreover, all heteroplasmic substitutions above the 2 % level were consistently detected with SBS one-channel chemistry. However, detection of low-level mtDNA variants may require additional, confirmatory experiments. In summary, the iSeq 100 system enables reproducible and accurate sequencing of human mitochondrial genomes. Detection of mtDNA minority variants depends on the laboratory protocol and sequencing platform used, but homoplasmic mutations and heteroplasmy above the 2 % level can be correctly detected with the iSeq 100 system.

Automated summary

The significance of mtDNA heteroplasmy in forensic and medical genetics has been heightened due to advancements in MPS technologies. This study aimed to assess the capabilities of the iSeq 100 sequencing system by sequencing previously determined mitochondrial genomes. The iSeq 100 system demonstrated accurate and reproducible sequencing of human mitochondrial genomes, detecting homoplasmic mutations and heteroplasmy above the 2% level.