Article ; Online: Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow.
2022 Volume 63, Issue 3, Page(s) 224–230
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.: ... ...
| 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 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). |
|---|---|
| MeSH term(s) | DNA, Mitochondrial/genetics ; Genome, Mitochondrial/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA/methods ; Workflow |
| Chemical Substances | DNA, Mitochondrial |
| Language | English |
| Publishing date | 2022-06-13 |
| Publishing country | Croatia |
| Document type | Journal Article |
| ZDB-ID | 1157623-6 |
| ISSN | 1332-8166 ; 0353-9504 |
| ISSN (online) | 1332-8166 |
| ISSN | 0353-9504 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
More links
Kategorien
In stock of ZB MED Cologne/Königswinter
| Zs.A 187: Show issues | Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG) |
Order via subito
This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.