Highly Accurate Sequence- and Position-Independent Error Profiling of DNA Synthesis and Sequencing

A comprehensive error analysis of DNA-stored data during processing, such as DNA synthesis and sequencing, is crucial for reliable DNA data storage. Both synthesis and sequencing errors depend on the sequence and the transition of bases of nucleotides; ignoring either one of the error sources leads to technical challenges in minimizing the error rate. Here, we present a methodology and toolkit that utilizes an oligonucleotide library generated from a 10-base-shifted sequence array, which is individually labeled with unique molecular identifiers, to delineate and profile DNA synthesis and sequencing errors simultaneously. This methodology enables position- and sequence-independent error profiling of both DNA synthesis and sequencing. Using this toolkit, we report base transitional errors in both synthesis and sequencing in general DNA data storage as well as degenerate-base-augmented DNA data storage. The methodology and data presented will contribute to the development of DNA sequence designs with minimal error.