A long-amplicon nanopore sequencing and analysis method for human whole mitochondrial genome.

Human mitochondrial DNA (mtDNA) has long been a pivotal niche in medical genetics, human forensics, and anthropology. However, common methods for mtDNA analysis are usually based on short-read sequencing and rely on read slicing of multiple PCR amplicons, which may be influenced by nuclear mitochondrial sequences (NUMTs) and cause confusion in identifying heteroplasmy. Nanopore sequencing, by contrast, theoretically enables direct, real-time analysis of nucleic acid fragments of any length, offering new insights into full-length sequencing and heterogeneity authentication of the entire mitochondrial genome (mitogenome). Here, we presented a single-amplicon long-read sequencing method for analysis of the whole mitogenome using the QNome nanopore sequencer (Qitan Technology). An open-source bioinformatics pipeline, VCall, was developed to automatically analyze the nanopore long-read sequencing data. Subsequently, 92 random samples were analyzed to characterize mtDNA polymorphisms, heteroplasmic sites, and complex sequences and positions using this approach. For two DNA standard materials (Control DNA 9947 A and 9948), the average read length exceeded 16,300 bp, with an average total read number of 10,499. For the random samples, after applying filtering criteria (read length: 15-17 kb; Q-score > 10), we obtained a mean of 4263 total reads and 150 Mb total bases per sample. Among the 92 samples, 85 haplotypes and 72 haplogroups were identified, with haplotype and haplogroup diversities of 0.9981 and 0.9936, respectively. Using a 10.0 % detection threshold, heteroplasmy was observed at 61 nucleotide positions, 17 of which contained minor bases not previously reported in the MITOMAP database. Before applying nanopore technology for routine detection and analysis of forensic mtDNA, heteroplasmic sites need to be thoroughly validated, and more population samples need to be further tested. Nevertheless, this study lays a solid foundation for the application of nanopore sequencing in mtDNA research, and the VCall pipeline developed herein is also applicable to other long-read sequencing platforms.