0324-8267 Wydawnictwo Uniwersytetu Jagiellońskiego Low-level point heteroplasmy detection in human mitogenomes amplified with different polymerases and sequenced on MiSeq FGx platform Wykrywanie heteroplazmii punktowej na niskim poziomie w ludzkich genomach mitochondrialnych amplifikowanych różnymi polimerazami i sekwencjonowanych przy użyciu platformy MiSeq FGx Skonieczna Katarzyna author Ciesielka Marzanna author Teresiński Grzegorz author Grzybowski Tomasz author Katedra Medycyny Sądowej, Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy, Uniwersytet Mikołaja Kopernika w Toruniu, Uniwersytet Medyczny w Lublinie 016f61126 Polskie Towarzystwo Medycyny Sądowej i Kryminologii Katedra Medycyny Sądowej, Zakład Genetyki Molekularnej i Sądowej, Wydział Lekarski, Collegium Medicum Uniwersytetu Mikołaja Kopernika Correspondence to: Katarzyna Skonieczna katarzyna.skonieczna@cm.umk.pl Correspondence to: Marzanna Ciesielka Correspondence to: Grzegorz Teresiński Grzegorz.Teresinski@umlub.edu.pl Correspondence to: Tomasz Grzybowski 11 12 2023 Vol. 73 (2) 2023 131 138 Copyright © 2023 2023 <p><strong>Introduction</strong>: Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied.</p> <p><strong><br />Aim</strong>: The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes.</p> <p><strong><br />Materials and Methods</strong>: Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level.</p> <p><strong><br />Results</strong>: All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine.</p> <p><strong><br />Conclusions</strong>: The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes.</p> 1 McElhoe JA, Wilton PR, Parson W, Holland MM. Exploring statistical weight estimates for mitochondrial DNA matches involving heteroplasmy. Int J Legal Med. 2022; 136: 671-685. 2 McCormick EM, Lott MT, Dulik MC, Shen L, Attimonelli M, Vitale O, Karaa A, Bai R, Pineda-Alvarez DE, Singh LN, Stanley CM, Wong S, Bhardwaj A, Merkurjev D, Mao R, Sondheimer N, Zhang S, Procaccio V, Wallace DC, Gai X, Falk MJ. Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation. Hum Mutat. 2020; 41: 2028-2057. 3 Fazzini F, Fendt L, Schönherr S, Forer L, Schöpf B, Streiter G, Losso JL, Kloss-Brandstätter A, Kronenberg F, Weissensteiner H. Analyzing Low-Level mtDNA Heteroplasmy-Pitfalls and Challenges from Bench to Benchmarking. Int J Mol Sci. 2021; 22: 935. 4 Marshall C, Parson W. Interpreting NUMTs in forensic genetics: Seeing the forest for the trees. Forensic Sci Int Genet. 2021; 53: 102497. 5 Skonieczna K, Malyarchuk B, Jawień A, Marszałek A, Banaszkiewicz Z, Jarmocik P, Grzybowski T. Mitogenomic differences between the normal and tumor cells of colorectal cancer patients. Hum Mutat. 2018; 39: 691-701. 6 Fendt, L, Zimmermann B, Daniaux M, Parson W. Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences. BMC Genomics 2009; 10: 139. 7 Skonieczna K, Grzybowski T. Capability of the iSeq 100 sequencing system from Illumina to detect low-level substitutions in the human mitochondrial genome. Forensic Sci Int Genet. 2023; 66: 102912. 8 Andrews S. FastQC: a quality control tool for high throughput sequence data. 2010; Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc 9 Weissensteiner H, Forer L, Fuchsberger C, Schöpf B, Kloss-Brandstätter A, Specht G, Kronenberg F, Schönherr S. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud. Nucleic Acids Res. 2016; 44: W64-W69. 10 McElhoe JA, Holland MM. Characterization of background noise in MiSeq MPS data when sequencing human mitochondrial DNA from various sample sources and library preparation methods. Mitochondrion. 2020; 52:40-55. 11 Holland MM, Wilson LA, Copeland S, Dimick G, Holland CA, Bever R, McElhoe JA. MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment. Int J Legal Med. 2017; 131: 919-931. 12 Kloss-Brandstätter A, Weissensteiner H, Erhart G, Schäfer G, Forer L, Schönherr S, Pacher D, Seifarth C, Stöckl A, Fendt L, Sottsas I, Klocker H, Huck CW, Rasse M, Kronenberg F, Kloss FR. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma. PLoS One. 2015; 10: e0135643. 13 Hestand MS, Van Houdt J, Cristofoli F, Vermeesch JR. Polymerase specific error rates and profiles identified by single molecule sequencing. Mutat Res. 2016; 784-785: 39-45. 14 McInerney P, Adams P, Hadi MZ. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Mol Biol Int. 2014;2014:287430. 15 Ring JD, Sturk-Andreaggi K, Peck MA, Marshall C. A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons. Forensic Sci Int Genet. 2017; 29:174-180. 16 Peck MA, Brandhagen MD, Marshall C, Diegoli TM, Irwin JA, Sturk-Andreaggi K. Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq. Forensic Sci Int Genet. 2016; 24:103-111.