OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands for genomics research and biotechnology applications. However, the same level of progress has not been achieved for shorter sequences, such as RNA transcripts and DNA oligonucleotides. Oligonucleotides play a crucial role in automated genome engineering through oligo library generation and in DNA data storage, where they encode binary information in DNA libraries. The accurate sequencing of large and diverse oligonucleotide libraries hence is essential, however, accessible sequencing solutions for oligonucleotides — particularly DNA primers for PCR, oligo DNA libraries for mutagenesis or RNA libraries used in gene expression analysis — remain inadequate. Current biological research platforms still rely on expensive and cumber-some quality control methods such as high-performance liquid chromatography and mass spectrometry. 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. 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.