Accurate sequencing of DNA motifs able to form alternative (non-B) structures.
Accurate sequencing of DNA motifs able to form alternative (non-B) structures.
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能够形成替代（非B）结构的DNA基序的精确测序。
DOI：
10.1101/gr.277490.122
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发表时间：
2023-06
期刊：
影响因子：
7
通讯作者：
Makova, Kateryna D.
中科院分区：
文献类型：
--
作者：
Weissensteiner, Matthias H.;Cremona, Marzia A.;Guiblet, Wilfried M.;Stoler, Nicholas;Harris, Robert S.;Cechova, Monika;Eckert, Kristin A.;Chiaromonte, Francesca;Huang, Yi-Fei;Makova, Kateryna D.
在某些基序上，大约13%的人类基因组有可能形成非规范(非B)DNA结构(如G-四链、十字形和Z-DNA)，这些结构调节许多细胞过程，但也影响聚合酶和解旋酶的活性。因为测序技术使用这些酶，它们可能在非B结构上具有更多的错误。为了评估这一点，我们分析了Illumina、太平洋生物科学(PacBio)HiFi和牛津纳米孔技术(ONT)在非B基序上的错误率、阅读深度和基本质量。所有技术都显示大多数非B基序类型的测序成功，尽管这可能是由于几个因素，包括结构形成、GC含量偏差和均聚物的存在。在所有非B基序类型中，单核苷酸错配误差在HiFi和ONT中的倾向性较低，但在所有三种技术中，G-四链和Z-DNA的单核苷酸错配误差均增加。在Illumina和HiFi中，除Z-DNA外，所有非B型的缺失错误均增加，在ONT中仅G-四链缺失。在Illumina、HiFi和ONT中，非B基序的插入错误分别是高度、中等和轻微增加的。此外，我们开发了一种概率方法来确定非B基序上的假阳性数量，这取决于样本大小和变异频率，并将其应用于公开可用的数据集(1000基因组、西蒙斯基因组多样性项目和gnomAD)。我们的结论是，在低阅读深度的研究(单细胞、古代DNA和混合样本群体测序)和对罕见变异进行评分时，应该考虑非B DNA基序上的测序错误。在未来对非B型DNA的研究中，联合技术应能最大限度地提高测序准确性。
Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.
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