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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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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
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author Sukser, Viktorija
Korolija, Marina
Račić, Ivana
Rožić, Sara
Barbarić, Lucija
author_facet Sukser, Viktorija
Korolija, Marina
Račić, Ivana
Rožić, Sara
Barbarić, Lucija
author_sort Sukser, Viktorija
collection PubMed
description 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).
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spelling pubmed-92840142022-07-29 Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow Sukser, Viktorija Korolija, Marina Račić, Ivana Rožić, Sara Barbarić, Lucija Croat Med J Research Article 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). Croatian Medical Schools 2022-06 /pmc/articles/PMC9284014/ /pubmed/35722691 http://dx.doi.org/10.3325/cmj.2022.63.224 Text en Copyright © 2022 by the Croatian Medical Journal. All rights reserved. https://creativecommons.org/licenses/by/2.5/This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Sukser, Viktorija
Korolija, Marina
Račić, Ivana
Rožić, Sara
Barbarić, Lucija
Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title_full Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title_fullStr Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title_full_unstemmed Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title_short Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
title_sort human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow
topic Research Article
url 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
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