Abstract
Non-B DNA structures formed by repetitive sequence motifs are known instigators of mutagenesis in experimental systems. Analyzing this phenomenon computationally in the human genome requires careful disentangling of intrinsic confounding factors, including overlapping and interrupted motifs and recurrent sequencing errors. Here, we show that accounting for these factors eliminates all signals of repeat-induced mutagenesis that extend beyond the motif boundary, and eliminates or dramatically shrinks the magnitude of mutagenesis within some motifs, contradicting previous reports. Mutagenesis not attributable to artifacts revealed several biological mechanisms. Polymerase slippage generates frequent indels within every variety of short tandem repeat motif, implicating slipped-strand structures. Interruption-correcting single nucleotide variants within short tandem repeats may originate from error-prone polymerases. Secondary-structure formation promotes single nucleotide variants within palindromic repeats and duplications within direct repeats. G-quadruplex motifs cause recurrent sequencing errors, whereas mutagenesis at Z-DNAs is conspicuously absent.
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Data availability
The datasets analyzed during this study are freely available from the gnomAD Consortium (https://gnomad.broadinstitute.org/downloads), the UCSC Genome Browser (https://genome.ucsc.edu), the non-B Database (https://nonb-abcc.ncifcrf.gov/apps/nBMST/default/) and other studies as cited. Instructions for accessing specific datasets are further detailed in code repository (Code availability).
Code availability
The code to perform the analysis in this study is available in a Github repository (https://github.com/ryanmcggg/nonb_motifs). For software/packages (with version numbers), please visit the Github repository.
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Acknowledgements
We thank V. Seplyarskiy and E. Koch for their important contributions. The project has been funded by National Institutes of Health grants R35-GM127131, 67 R01-MH101244, U01-HG012009 and R01-HG010372 (awarded to S.S.).
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R.M. conceived the study, performed the analysis and wrote the manuscript. S.S. supervised the project, discussed results and consulted on the manuscript.
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Extended data
Extended Data Fig. 1 Caveats of the ‘Non-B Database’.
a, Overlapping motifs in Non-B DB. X-axis indicates motif category. Y-axis indicates the portion of motifs that do not overlap other categories (‘unique’) and those that overlap additional categories (indicated by color and abbreviation). b, Overlapping flanking regions in Non-B DB. For each motif in Non-B DB, the distance to the nearest repeat (including transposable elements from Repeatmasker where indicated) on either side was measured, and the smaller of the upstream or downstream values was taken. Overlapping motifs have a distance of 0. X-axis represents the percentile for the range of distances, and the Y-axis indicates the distance in nucleotides.
Extended Data Fig. 2 Mutagenesis flanking non-B motifs.
X-axes are coordinates relative to central motif (0 position encompasses entire repeat). Y-axes are relative mutation frequency (compared to the gnomAD average, normalized by trinucleotide mutation type). Data presented as mean values, with 95% binomial confidence intervals indicated in transparency. Confidence values derived from n = 76,156 individuals and the dynamic loci count. Blue lines: no sequencing quality filters, green and yellow lines: increasingly stringent filters (‘pass’ indicates gnomAD’s passing quality filter based on a VQSLOD score of −2.774). Left: STR motifs (combined with their respective reverse complements). Right: Other non-B motifs. STRs and Symmetrical motifs exclude the shortest 80% by motif length. G4 motifs (strand-specific) detected under K + conditions. Z-DNA motifs detected using standard definition. See Supplementary Fig. 2a–c and Methods for more details.
Extended Data Fig. 3 Absolute mutation frequency contributing to STR interruption reversions.
Mutation frequencies within STRs, represented as absolute frequencies (mutations per base). Frequency of interruption-perfecting SNVs (left), insertions within imperfect motifs (middle), and deletion of imperfections (right). X-axes are motif lengths. Y-axes are relative mutation frequency. Data presented as mean values, with error bars indicating 95% binomial confidence intervals. Confidence values derived from n = 76,156 individuals and the dynamic loci count. Blue: no sequencing quality filters, green and yellow: increasingly stringent quality filters. Motif sequence (combined with its reverse-complement) indicated at left of each row.
Extended Data Fig. 4 Duplications within direct repeats.
a, Examples of duplications within direct repeats. Reference sequence shown. Blue text indicates location of repeat motifs. Blue highlight shows location of motif mismatches. Orange highlight indicates region duplicated in gnomAD. Orange text with blue highlight indicates that the duplication includes the interruption. b, Duplication position bias. Duplication start/end positions (blue) or positions flanking duplications (orange), categorized by their location (ie. start of left motif, mismatch position in left motif, end of spacer, etc.). Frequency of the duplication at each position (observed) is divided by the portion of the motif for which the position accounts (expected). Represents n = 3170 DR loci with spacer length < 10nt and containing a duplication >5 nt. c) Gap repair model explaining direct repeat duplications. The initial A-B-A pattern forms a slipped-strand structure. Post-replicative gap-filling (Polζ) fills in single-stranded loop regions, resulting in 4-way pseudo-Holliday junctions. Cleavage of the top strand allows the filled-in sequence to be ligated into the top strand. Either this can be repeated for the other loop, or replication of the top strand will produce a daughter cell with the A-B-A-B-A pattern.
Extended Data Fig. 5 G4 motifs are prone to recurrent sequencing errors.
a, Insertions and b, deletions within G4 motifs. X-axis indicates positions within G-runs, or spacers between G-runs. Spacers of 1 nt in length are categorized separately. Y-axis is relative mutation frequency. Data presented as mean values, with error bars indicating 95% binomial confidence intervals. Confidence values derived from n = 76,156 individuals and the dynamic loci count. Blue: no sequencing quality filters, green and yellow: increasingly stringent filters. Arrows indicate magnitude and direction of change for individual trinucleotide mutation frequencies before and after sequencing quality filters. Mutations with large magnitude changes are highlighted in text.
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Supplementary Figs. 1–7.
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McGinty, R.J., Sunyaev, S.R. Revisiting mutagenesis at non-B DNA motifs in the human genome. Nat Struct Mol Biol 30, 417–424 (2023). https://doi.org/10.1038/s41594-023-00936-6
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DOI: https://doi.org/10.1038/s41594-023-00936-6
