engleski

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

Aim: Optimized and efficient library preparation workflow is one of the most important prerequisites for obtaining high quality results in massively parallel sequencing (MPS). Critical steps in Illumina® Human mtDNA Genome assay include target enrichment (long-range PCR of mtDNA in two fragments), limited-cycle PCR (addition of index-adapters), and library normalization. We aimed to test and evaluate those three steps in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples. Methods: Three long-range high-fidelity DNA polymerases (PlatinumTM PCR SuperMix High Fidelity, LA Taq® Hot Start and PrimeSTAR® GXL) were tested for their performance in 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. Also, two library normalization methods were compared: bead-based normalization versus 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 impact either 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 MPS workflows (e.g. small genomes, PCR amplicons and plasmids).

MiSeq, mitochondrial DNA, Nextera XT, optimization, small genome sequencing

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