The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

  • Anton Shikov Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation; All-Russia Research Institute for Agricultural Microbiology, Shosse Podbel'skogo, 3, Saint Petersburg, 190608, Russian Federation; Faculty of Medicine, Saint Petersburg State University, 21-ya liniya, 8a, Saint Petersburg, 199106, Russian Federation
  • Viktoriya Tsay Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation
  • Mikhail Fedyakov Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation
  • Yuri Eismont Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation
  • Alena Rudnik Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation
  • Stanislav Urasov Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation
  • Sergey Sherbak Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation; Faculty of Medicine, Saint Petersburg State University, 21-ya liniya, 8a, Saint Petersburg, 199106, Russian Federation
  • Oleg Glotov Genetics Laboratory, City Hospital No. 40, ul. Borisova, 9, Saint Petersburg, 197706, Russian Federation; Department of Genomic Medicine, D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology, Mendeleyevskaya liniya, 3, Saint Petersburg, 199034, Russian Federation


The emergence of long-read sequencing technologies has made a revolutionary step in genome biology and medicine. However, long reads are characterized by a relatively high error rate, impairing their usage for variant calling as a part of routine practice. Thus, we here examine different popular variant callers on long-read sequences of the human mitochondrial genome, convenient in terms of small size and easily obtained high coverage. The sequencing of mitochondrial DNA from 8 patients was conducted via Illumina (MiSeq) and the Oxford Nanopore platform (MinION), with the former utilized as a gold standard when evaluating variant calling’s accuracy. We used a conventional GATK3-BWA-based pipeline for paired-end reads and Guppy basecaller coupled with minimap2 for MinION data, respectively. We then compared the outputs of Clairvoyante, Nanopolish, GATK3, Longshot, DeepVariant, and Varscan tools applied on long-read alignments by analyzing false-positive and false-negative rates. While for most callers, raw signals represented false positives due to homopolymeric errors, Nanopolish demonstrated both high similarity (Jaccard coefficient of 0.82) and a comparable number of calls with the Illumina data (140 vs. 154) with the best performance according to AUC (area under ROC curve, 0.953) as well. In sum, our results, despite being obtained from a small dataset, provide evidence that sufficient coverage coupled with an optimal pipeline could make long reads of mitochondrial DNA applicable for variant calling.


next-generation sequencing, Oxford Nanopore, Illumina, variant calling, mitochondrial DNA


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How to Cite
Shikov, A., Tsay, V., Fedyakov, M., Eismont, Y., Rudnik, A., Urasov, S., Sherbak, S., & Glotov, O. (2021). The application of Nanopore sequencing for variant calling on the human mitochondrial DNA. Biological Communications, 66(2), 109–123.
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