Analyzing Low-Level mtDNA Heteroplasmy—Pitfalls and Challenges from Bench to Benchmarking
| العنوان: | Analyzing Low-Level mtDNA Heteroplasmy—Pitfalls and Challenges from Bench to Benchmarking |
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| المؤلفون: | Hansi Weissensteiner, Federica Fazzini, Anita Kloss-Brandstätter, Bernd Schöpf, Florian Kronenberg, Jamie Lee Losso, Lukas Forer, Gertraud Streiter, Liane Fendt, Sebastian Schönherr |
| المصدر: | International Journal of Molecular Sciences Volume 22 Issue 2 International Journal of Molecular Sciences, Vol 22, Iss 935, p 935 (2021) |
| بيانات النشر: | MDPI AG, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | 0301 basic medicine, Aging, Mitochondrial DNA, DNA polymerase, DNA-Directed DNA Polymerase, next generation sequencing (NGS), Computational biology, DNA, Mitochondrial, Article, Catalysis, lcsh:Chemistry, Inorganic Chemistry, 03 medical and health sciences, 0302 clinical medicine, Humans, Genetic Predisposition to Disease, heteroplasmy, Physical and Theoretical Chemistry, lcsh:QH301-705.5, Molecular Biology, Spectroscopy, Polymerase, variant callers, Massive parallel sequencing, biology, Organic Chemistry, Genetic Variation, High-Throughput Nucleotide Sequencing, Sequence Analysis, DNA, General Medicine, Variant allele, Benchmarking, mitochondrial DNA (mtDNA), DNA extraction, Heteroplasmy, Mitochondria, Computer Science Applications, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, 030220 oncology & carcinogenesis, Genome, Mitochondrial, Mutation, biology.protein |
| الوصف: | Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging. |
| وصف الملف: | application/pdf |
| تدمد: | 1422-0067 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::197f37d81a1d38131be454c7697ef271 https://doi.org/10.3390/ijms22020935 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....197f37d81a1d38131be454c7697ef271 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14220067 |
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