Izvorni znanstveni članak
https://doi.org/10.3325/cmj.2022.63.224
Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
Viktorija Sukser
; Department of Biology and Fibers, Forensic Science Center “Ivan Vučetić”, Zagreb, Croatia
Marina Korolija
; Department of Biology and Fibers, Forensic Science Center “Ivan Vučetić”, Zagreb, Croatia
Ivana Račić
; Department of Biology and Fibers, Forensic Science Center “Ivan Vučetić”, Zagreb, Croatia
Sara Rožić
; Department of Biology and Fibers, Forensic Science Center “Ivan Vučetić”, Zagreb, Croatia
Lucija Barbarić
; Department of Biology and Fibers, Forensic Science Center “Ivan Vučetić”, Zagreb, Croatia
Sažetak
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 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).
Ključne riječi
Hrčak ID:
290149
URI
Datum izdavanja:
23.6.2022.
Posjeta: 573 *