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Discrimination of monozygotic twins using mtDNA heteroplasmy through probe capture enrichment and massively parallel sequencing

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Abstract

Differentiating between monozygotic (MZ) twins remains difficult because they have the same genetic makeup. Applying the traditional STR genotyping approach cannot differentiate one from the other. Heteroplasmy refers to the presence of two or more different mtDNA copies within a single cell and this phenomenon is common in humans. The levels of heteroplasmy cannot change dramatically during transmission in the female germ line but increase or decrease during germ-line transmission and in somatic tissues during life. As massively parallel sequencing (MPS) technology has advanced, it has shown the extraordinary quantity of mtDNA heteroplasmy in humans. In this study, a probe hybridization technique was used to obtain mtDNA and then MPS was performed with an average sequencing depth of above 4000. The results showed us that all ten pairs of MZ twins were clearly differentiated with the minor heteroplasmy threshold at 1.0%, 0.5%, and 0.1%, respectively. Finally, we used a probe that targeted mtDNA to boost sequencing depth without interfering with nuclear DNA and this technique can be used in forensic genetics to differentiate the MZ twins.

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References

  1. Oosthuizen T, Howes LM (2022) The development of forensic DNA analysis: new debates on the issue of fundamental human rights. Forensic Sci Int Genet 56:102606

    Article  CAS  PubMed  Google Scholar 

  2. Nwawuba Stanley U et al (2020) Forensic DNA profiling: autosomal short tandem repeat as a prominent marker in crime investigation. Malays J Med Sci 27(4):22–35

    PubMed  PubMed Central  Google Scholar 

  3. Xu QN, Li CT, Liu XL (2018) Research progress on discrimination of monozygotic twins. Fa Yi Xue Za Zhi 34(6):672–677

    CAS  PubMed  Google Scholar 

  4. Yang YR et al (2012) Progress on epigenetics applications in forensic science. Fa Yi Xue Za Zhi 28(5):366–370

    CAS  PubMed  Google Scholar 

  5. Vidaki A, Daniel B, Court DS (2013) Forensic DNA methylation profiling--potential opportunities and challenges. Forensic Sci Int Genet 7(5):499–507

    Article  CAS  PubMed  Google Scholar 

  6. Marqueta-Gracia JJ et al (2018) Differentially methylated CpG regions analyzed by PCR-high resolution melting for monozygotic twin pair discrimination. Forensic Sci Int Genet 37:e1–e5

    Article  CAS  PubMed  Google Scholar 

  7. Castellani CA et al (2014) Biological relevance of CNV calling methods using familial relatedness including monozygotic twins. BMC Bioinformatics 15:114

    Article  PubMed  PubMed Central  Google Scholar 

  8. Planterose Jimenez B et al (2021) Equivalent DNA methylation variation between monozygotic co-twins and unrelated individuals reveals universal epigenetic inter-individual dissimilarity. Genome Biol 22(1):18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xiao C et al (2019) Differences of microRNA expression profiles between monozygotic twins’ blood samples. Forensic Sci Int Genet 41:152–158

    Article  CAS  PubMed  Google Scholar 

  10. Nistico L et al (2006) Concordance, disease progression, and heritability of coeliac disease in Italian twins. Gut 55(6):803–808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sites ER et al (2017) Analysis of copy number variants in 11 pairs of monozygotic twins with neurofibromatosis type 1. Am J Med Genet A 173(3):647–653

    Article  CAS  PubMed  Google Scholar 

  12. Stewart JB, Chinnery PF (2021) Extreme heterogeneity of human mitochondrial DNA from organelles to populations. Nat Rev Genet 22(2):106–118

    Article  CAS  PubMed  Google Scholar 

  13. Anderson S et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290(5806):457–465

    Article  CAS  PubMed  Google Scholar 

  14. Chiaratti MR, Chinnery PF (2022) Modulating mitochondrial DNA mutations: factors shaping heteroplasmy in the germ line and somatic cells. Pharmacol Res 185:106466

    Article  CAS  PubMed  Google Scholar 

  15. Uchiumi T, Kang D (2012) The role of TFAM-associated proteins in mitochondrial RNA metabolism. Biochim Biophys Acta 1820(5):565–570

    Article  CAS  PubMed  Google Scholar 

  16. Yasukawa T, Kang D (2018) An overview of mammalian mitochondrial DNA replication mechanisms. J Biochem 164(3):183–193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kennedy SR et al (2013) Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. PLoS Genet 9(9):e1003794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Legati A et al (2021) Current and new next-generation sequencing approaches to study mitochondrial DNA. J Mol Diagn 23(6):732–741

    Article  CAS  PubMed  Google Scholar 

  19. Mertens J et al (2019) Detection of heteroplasmic variants in the mitochondrial genome through massive parallel sequencing. Bio Protoc 9(13):e3283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang Z et al (2015) Differentiating between monozygotic twins through next-generation mitochondrial genome sequencing. Anal Biochem 490:1–6

    Article  CAS  PubMed  Google Scholar 

  21. McElhoe JA et al (2014) Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq. Forensic Sci Int Genet 13:20–29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yu H et al (2022) Hot spots-making directed evolution easier. Biotechnol Adv 56:107926

    Article  PubMed  Google Scholar 

  23. Chen L et al (2020) Highly accurate mtGenome haplotypes from long-read SMRT sequencing can distinguish between monozygotic twins. Forensic Sci Int Genet 47:102306

    Article  CAS  PubMed  Google Scholar 

  24. Gaudin M, Desnues C (2018) Hybrid capture-based next generation sequencing and its application to human infectious diseases. Front Microbiol 9:2924

    Article  PubMed  PubMed Central  Google Scholar 

  25. Shih, S.Y., et al., Correction: Shelly Y. Shih; et al.; Applications of probe capture enrichment next generation sequencing for whole mitochondrial genome and 426 nuclear SNPs for forensically challenging samples. Genes 2018, 9, 49.

  26. Yin L et al (2021) Validation of the Microreader 28A ID System: A 6-dye multiplex amplification assay for forensic application. Electrophoresis 42(19):1928–1935

    Article  CAS  PubMed  Google Scholar 

  27. Shih SY et al (2018) Applications of probe capture enrichment next generation sequencing for whole mitochondrial genome and 426 nuclear SNPs for forensically challenging samples. Genes (Basel) 9(1)

  28. Lang J et al (2021) Evaluation of the MGISEQ-2000 sequencing platform for illumina target capture sequencing libraries. Front Genet 12:730519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chen S et al (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34(17):i884–i890

    Article  PubMed  PubMed Central  Google Scholar 

  30. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. McKenna A et al (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20(9):1297–1303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li H et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  33. Yang H, Wang K (2015) Genomic variant annotation and prioritization with ANNOVAR and wANNOVAR. Nat Protoc 10(10):1556–1566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bensasson D et al (2001) Mitochondrial pseudogenes: evolution’s misplaced witnesses. Trends Ecol Evol 16(6):314–321

    Article  CAS  PubMed  Google Scholar 

  35. Parakatselaki ME, Ladoukakis ED (2021) mtDNA heteroplasmy: origin, detection, significance, and evolutionary consequences. Life (Basel) 11(7):633

    CAS  PubMed  Google Scholar 

  36. Vidaki A et al (2017) Epigenetic discrimination of identical twins from blood under the forensic scenario. Forensic Sci Int Genet 31:67–80

    Article  CAS  PubMed  Google Scholar 

  37. Xu J et al (2015) LINE-1 DNA methylation: a potential forensic marker for discriminating monozygotic twins. Forensic Sci Int Genet 19:136–145

    Article  CAS  PubMed  Google Scholar 

  38. Zhang N et al (2015) Intra-monozygotic twin pair discordance and longitudinal variation of whole-genome scale DNA methylation in adults. PLoS One 10(8):e0135022

    Article  PubMed  PubMed Central  Google Scholar 

  39. Vidaki A et al (2018) Investigating the epigenetic discrimination of identical twins using buccal swabs, saliva, and cigarette butts in the forensic setting. Genes (Basel) 9(5):252

    Article  PubMed  PubMed Central  Google Scholar 

  40. Fang C et al (2019) MicroRNA profile analysis for discrimination of monozygotic twins using massively parallel sequencing and real-time PCR. Forensic Sci Int Genet 38:23–31

    Article  CAS  PubMed  Google Scholar 

  41. Li D et al (2022) Advances in bioactivity of MicroRNAs of plant-derived exosome-like nanoparticles and milk-derived extracellular vesicles. J Agric Food Chem 70(21):6285–6299

    Article  CAS  PubMed  Google Scholar 

  42. Liang H et al (2014) The origin, function, and diagnostic potential of extracellular microRNAs in human body fluids. Wiley Interdiscip Rev RNA 5(2):285–300

    Article  CAS  PubMed  Google Scholar 

  43. Rolf B, Krawczak M (2021) The germlines of male monozygotic (MZ) twins: Very similar, but not identical. Forensic Sci Int Genet 50:102408

    Article  CAS  PubMed  Google Scholar 

  44. Burr SP, Chinnery PF (2022) Measuring single-cell mitochondrial DNA copy number and heteroplasmy using digital droplet polymerase chain reaction. J Vis Exp 185:e63870

    Google Scholar 

  45. Abd Radzak SM et al (2022) Insights regarding mitochondrial DNA copy number alterations in human cancer (Review). Int J Mol Med 50(2):1–8

    Article  Google Scholar 

  46. Wang Y et al (2022) Genetic landscape of human mitochondrial genome using whole-genome sequencing. Hum Mol Genet 31(11):1747–1761

    Article  CAS  PubMed  Google Scholar 

  47. Dulovic-Mahlow M et al (2021) Discordant monozygotic parkinson disease twins: role of mitochondrial integrity. Ann Neurol 89(1):158–164

    Article  CAS  PubMed  Google Scholar 

  48. Yin D et al (2022) Gapless genome assembly of East Asian finless porpoise. Sci Data 9(1):765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sun Y et al (2021) Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders. BMC Med Genomics 14(1):102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lee J, Jung J (2022) First record of the complete mitochondrial genome of Tubifex tubifex (Muller) 1774 (Annelida; Clitellata; Oligochaeta) and phylogenetic analysis. Mitochondrial DNA B Resour 7(7):1208–1210

    Article  PubMed  PubMed Central  Google Scholar 

  51. Jeon SA et al (2021) Comparison between MGI and Illumina sequencing platforms for whole genome sequencing. Genes Genomics 43(7):713–724

    Article  CAS  PubMed  Google Scholar 

  52. Zhu K et al (2021) Comparative Performance of the MGISEQ-2000 and Illumina X-Ten Sequencing Platforms for Paleogenomics. Front Genet 12:745508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Korostin D et al (2020) Comparative analysis of novel MGISEQ-2000 sequencing platform vs Illumina HiSeq 2500 for whole-genome sequencing. PLoS One 15(3):e0230301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bardan F, Higgins D, Austin JJ (2023) A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry, hair and eye colour, and Y chromosome lineage. Forensic Sci Int Genet 63:102822

    Article  CAS  PubMed  Google Scholar 

  55. Bose N et al (2018) Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples. Forensic Sci Int Genet 34:186–196

    Article  CAS  PubMed  Google Scholar 

  56. Tu J et al (2015) Systematic characteristic exploration of the chimeras generated in multiple displacement amplification through next generation sequencing data reanalysis. PLoS One 10(10):e0139857

    Article  PubMed  PubMed Central  Google Scholar 

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Data availability

The data sets used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Funding

This study was supported by the Natural Science Foundation of Liaoning Province (2022-YGJC-45) and the Student Innovation and Entrepreneurship Training Program of China Medical University in 2023.

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Authors and Affiliations

  1. School of Forensic Medicine, China Medical University, No.77, Puhe Road, Shenbei New District, Shenyang, 110122, People’s Republic of China

    Yang Zhong, Yu-Zhang Li, Xi-kai Hou, Ying Pan, Ang Li, Xiu-mei Zhu, Peng Lv, Zhe Du & Jun Yao

  2. Key Laboratory of Forensic Bio-evidence Sciences, Shenyang, Liaoning Province, China

    Yang Zhong, Yu-Zhang Li, Xi-kai Hou, Ying Pan, Ang Li, Xiu-mei Zhu, Peng Lv, Zhe Du & Jun Yao

  3. China Medical University Center of Forensic Investigation, Chengdu, China

    Yang Zhong, Yu-Zhang Li, Xi-kai Hou, Ying Pan, Ang Li, Xiu-mei Zhu, Peng Lv, Zhe Du & Jun Yao

  4. Institute of Evidence Law and Forensic Science, China University of Political Science and Law, Beijing, China

    Kuo Zeng

  5. Department of Forensic Sciences, College of Criminal Justice, Naif University of Security Sciences, Riyadh, 11452, Kingdom of Saudi Arabia

    Atif Adnan

  6. Department of Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, China

    Ying Yang

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  1. Yang Zhong
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  2. Kuo Zeng
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  7. Ang Li
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  11. Ying Yang
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  12. Jun Yao
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Corresponding authors

Correspondence to Ying Yang or Jun Yao.

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Ethics approval

This study was approved by the Ethics Committee of China Medical University (Shenyang, Liaoning Province, People’s Republic of China).

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The authors declare that they have no competing interest.

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Yang Zhong, Kuo Zeng, and Atif Adnan are the co-first authors.

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Zhong, Y., Zeng, K., Adnan, A. et al. Discrimination of monozygotic twins using mtDNA heteroplasmy through probe capture enrichment and massively parallel sequencing. Int J Legal Med 137, 1337–1345 (2023). https://doi.org/10.1007/s00414-023-03033-x

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