Reference : Benchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Life sciences : Genetics & genetic processes
Systems Biomedicine
Benchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA
Lüth, Theresa [> >]
Schaake, Susen [> >]
Grünewald, Anne mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Molecular and Functional Neurobiology]
May, Patrick mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > Bioinformatics Core]
Trinh, Joanne [> >]
Weissensteiner, Hansi [> >]
Frontiers in Genetics
Yes (verified by ORBilu)
[en] nanopore sequencing ; long-read sequencing ; mtDNA ; mixtures ; benchmarking ; heteroplasmy ; haplogroups ; low-frequency variant ; haplogroups
[en] Background: Sequencing quality has improved over the last decade for long-reads, allowing for more accurate detection of somatic low-frequency variants. In this study, we used mixtures of mitochondrial samples with different haplogroups (i.e., a specific set of mitochondrial variants) to investigate the applicability of nanopore sequencing for low-frequency single nucleotide variant detection.Methods: We investigated the impact of base-calling, alignment/mapping, quality control steps, and variant calling by comparing the results to a previously derived short-read gold standard generated on the Illumina NextSeq. For nanopore sequencing, six mixtures of four different haplotypes were prepared, allowing us to reliably check for expected variants at the predefined 5%, 2%, and 1% mixture levels. We used two different versions of Guppy for base-calling, two aligners (i.e., Minimap2 and Ngmlr), and three variant callers (i.e., Mutserve2, Freebayes, and Nanopanel2) to compare low-frequency variants. We used F<sub>1</sub> score measurements to assess the performance of variant calling.Results: We observed a mean read length of 11 kb and a mean overall read quality of 15. Ngmlr showed not only higher F<sub>1</sub> scores but also higher allele frequencies (AF) of false-positive calls across the mixtures (mean F<sub>1</sub> score = 0.83; false-positive allele frequencies < 0.17) compared to Minimap2 (mean F<sub>1</sub> score = 0.82; false-positive AF < 0.06). Mutserve2 had the highest F<sub>1</sub> scores (5% level: F<sub>1</sub> score >0.99, 2% level: F<sub>1</sub> score >0.54, and 1% level: F<sub>1</sub> score >0.70) across all callers and mixture levels.Conclusion: We here present the benchmarking for low-frequency variant calling with nanopore sequencing by identifying current limitations.
Luxembourg Centre for Systems Biomedicine (LCSB): Bioinformatics Core (R. Schneider Group)
Fonds National de la Recherche - FnR
Researchers ; Professionals
FnR ; FNR11250962 > Anne Grünewald > ProtectMove > Reduced Penetrance In Hereditary Movement Disorders: Elucidating Mechanisms Of Endogenous Disease Protection P1: Markers And Mechanisms Of Reduced Penetrance In Lrrk2 Mutation Carriers > 01/01/2017 > 30/06/2020 > 2016

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