Nucleic acid microarrays are the only tools that can supply very large oligonucleotide libraries, cornerstones of the nascent fields of de novo gene assembly and DNA data storage. Although the chemical synthesis of oligonucleotides is highly developed and robust, it is not error free, requiring the design of methods that can correct or compensate for errors, or select for high-fidelity oligomers. However, outside the realm of array manufacturers, little is known about the sources of errors and their extent. In this study, we look at the error rate of DNA libraries synthesized by photolithography and dissect the proportion of deletion, insertion and substitution errors. We find that the deletion rate is governed by the photolysis yield. We identify the most important substitution error and correlate it to phosphoramidite coupling. Besides synthetic failures originating from the coupling cycle, we uncover the role of imperfections and limitations related to optics, highlight the importance of absorbing UV light to avoid internal reflections and chart the dependence of error rate on both position on the array and position within individual oligonucleotides. Being able to precisely quantify all types of errors will allow for optimal choice of fabrication parameters and array design.
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.