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Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow

AIM: To evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples. METHODS: Three long-range high-fidelity DNA polymerases...

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Detalles Bibliográficos
Autores principales: Sukser, Viktorija, Korolija, Marina, Račić, Ivana, Rožić, Sara, Barbarić, Lucija
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Croatian Medical Schools 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9284014/
https://www.ncbi.nlm.nih.gov/pubmed/35722691
http://dx.doi.org/10.3325/cmj.2022.63.224
Descripción
Sumario:AIM: To evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples. METHODS: Three long-range high-fidelity DNA polymerases (Platinum(TM) PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization. RESULTS: PrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization. CONCLUSION: Optimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).