HiFi long-read amplicon sequencing for full-spectrum variants of human mtDNA.
BMC Genomics
; 25(1): 538, 2024 May 31.
Article
en En
| MEDLINE
| ID: mdl-38822239
ABSTRACT
BACKGROUND:
Mitochondrial diseases (MDs) can be caused by single nucleotide variants (SNVs) and structural variants (SVs) in the mitochondrial genome (mtDNA). Presently, identifying deletions in small to medium-sized fragments and accurately detecting low-percentage variants remains challenging due to the limitations of next-generation sequencing (NGS).METHODS:
In this study, we integrated targeted long-range polymerase chain reaction (LR-PCR) and PacBio HiFi sequencing to analyze 34 participants, including 28 patients and 6 controls. Of these, 17 samples were subjected to both targeted LR-PCR and to compare the mtDNA variant detection efficacy.RESULTS:
Among the 28 patients tested by long-read sequencing (LRS), 2 patients were found positive for the m.3243 A > G hotspot variant, and 20 patients exhibited single or multiple deletion variants with a proportion exceeding 4%. Comparison between the results of LRS and NGS revealed that both methods exhibited similar efficacy in detecting SNVs exceeding 5%. However, LRS outperformed NGS in detecting SNVs with a ratio below 5%. As for SVs, LRS identified single or multiple deletions in 13 out of 17 cases, whereas NGS only detected single deletions in 8 cases. Furthermore, deletions identified by LRS were validated by Sanger sequencing and quantified in single muscle fibers using real-time PCR. Notably, LRS also effectively and accurately identified secondary mtDNA deletions in idiopathic inflammatory myopathies (IIMs).CONCLUSIONS:
LRS outperforms NGS in detecting various types of SNVs and SVs in mtDNA, including those with low frequencies. Our research is a significant advancement in medical comprehension and will provide profound insights into genetics.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN Mitocondrial
/
Enfermedades Mitocondriales
/
Secuenciación de Nucleótidos de Alto Rendimiento
Límite:
Adult
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
BMC Genomics
Asunto de la revista:
GENETICA
Año:
2024
Tipo del documento:
Article
País de afiliación:
China