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

ABSTRACT:

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 (PlatinumTM 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 performe

SUBMITTER: Sukser V 

PROVIDER: S-EPMC9284014 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature