Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization.
Clin Chem
; 67(7): 968-976, 2021 07 06.
Article
em En
| MEDLINE
| ID: mdl-33822904
ABSTRACT
BACKGROUND:
The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification.METHODS:
We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%.RESULTS:
A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow.CONCLUSION:
dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Mitocondrial
/
Sequenciamento de Nucleotídeos em Larga Escala
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article