OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g. over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information (such as binary code) in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides — particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis — remain inadequate. To address this gap, we develop OligoSeq , an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation of either sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥90 % identity spanning from about 70 % to 10 % with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq uses nanopore technology and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.