Background Chemically synthesized oligonucleotides are vital to most nucleic acids-based technologies and several applications are sensitive to oligonucleotide sequence errors. However, it is challenging to identify and quantify the types and amount of errors in synthetic oligonucleotides. Methods We applied a digital sequencing approach using unique molecular identifiers to quantify errors in chemically synthesized oligonucleotides from multiple manufacturers with different synthesis strategies, purity grades, batches, and sequence context. Results We detected both deletions and substitutions in chemical oligonucleotide synthesis, but deletions were 7 times more common. We found that 97.2% of all analyzed oligonucleotide molecules were intact across all manufacturers and purity grades, although the number of oligonucleotide molecules with deletions ranged between 0.2% and 11.7% for different types. Different batches of otherwise identical oligonucleotide types also varied significantly, and batch effect can impact oligonucleotide quality more than purification. We observed a bias of increased deletion rates in chemically synthesized oligonucleotides toward the 5’-end for 1 out of 2 sequence configurations. We also demonstrated that the performance of sequencing assays depends on oligonucleotide quality. Conclusions Our data demonstrate that manufacturer, synthesis strategy, purity, batch, and sequence context all contribute to errors in chemically synthesized oligonucleotides and need to be considered when choosing and evaluating oligonucleotides. High-performance oligonucleotides are essential in numerous molecular applications, including clinical diagnostics.

中文翻译：

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化学寡核苷酸合成错误的数字量化

背景化学合成的寡核苷酸对大多数基于核酸的技术至关重要，并且一些应用对寡核苷酸序列错误很敏感。然而，识别和量化合成寡核苷酸中错误的类型和数量具有挑战性。方法 我们应用了一种数字测序方法，使用独特的分子标识符来量化来自多个制造商的化学合成寡核苷酸中的错误，这些制造商具有不同的合成策略、纯度等级、批次和序列背景。结果 我们在化学寡核苷酸合成中检测到缺失和取代，但缺失的发生率高出 7 倍。我们发现所有分析的寡核苷酸分子中有 97.2% 在所有制造商和纯度等级中都是完整的，尽管不同类型的缺失寡核苷酸分子的数量在 0.2% 到 11.7% 之间。不同批次的其他相同寡核苷酸类型也有显着差异，批次效应对寡核苷酸质量的影响大于纯化。我们观察到在化学合成的寡核苷酸中，对于 2 种序列配置中的 1 种，缺失率的增加偏向 5' 端。我们还证明了测序分析的性能取决于寡核苷酸的质量。结论 我们的数据表明，制造商、合成策略、纯度、批次和序列背景都会导致化学合成寡核苷酸的错误，在选择和评估寡核苷酸时需要加以考虑。