Zero-mode waveguides and nanopore-based sequencing technologies accelerate single-molecule studies

Abstract

Single-molecule technologies can provide detailed information regarding molecular mechanisms and interactions that cannot easily be studied on the bulk scale; generally, individual molecular behaviors cannot be distinguished, and only average characteristics can be measured. Nevertheless, the development of the single-molecule sequencer had a significant impact on conventional in vitro single-molecule research, featuring automated equipment, high-throughput chips, and automated analysis systems. However, the utilization of sequencing technology in in vitro single-molecule research is not yet globally prevalent, owing to the large gap between highly organized single-molecule sequencing and manual-based in vitro single-molecule research. Here, we describe the principles of zero-mode waveguides (ZMWs) and nanopore methods used as single-molecule DNA sequencing techniques, and provide examples of functional biological measurements beyond DNA sequencing that contribute to a global understanding of the current applications of these sequencing technologies. Furthermore, through a comparison of these two technologies, we discuss future applications of DNA sequencing technologies in in vitro single-molecule research.

Od Sangera do sekwencjonowania genomów – przegląd technologii sekwencjonowania DNA

“…ZMW (ang. Zero-Mode Waveguides) [51] znajdujących się na płytce do sekwencjonowania (SMRT Cell). Na dnie studzienek znajdują się miniaturowe otwory, przez które wpada światło.…”

A machine-readable specification for genomics assays

“…Illumina sequencing employs a sequencing by synthesis approach where fluorescently labeled nucleotides are incorporated into single-stranded DNA, and imaged, while PacBio uses zero-mode waveguides for single-molecule detection of dNTP incorporation. Oxford Nanopore on the other hand binds sequencing adapters to pores in a flow cell and DNA is sequenced by changes in electrical resistance across the pore ( Iizuka et al 2022 ).…”

A machine-readable specification for genomics assays

“…Illumina sequencing employs a sequencing by synthesis approach where fluorescently labeled nucleotides are incorporated into single-stranded DNA, and imaged, while PacBio uses zero-mode waveguides for single-molecule detection of dNTP incorporation. Oxford Nanopore on the other hand binds sequencing adapters to pores in a flow cell and DNA is sequenced by changes in electrical resistance across the pore (Iizuka, Yamazaki, and Uemura 2022). Many single-cell genomics assays introduce additional library complexity further complicating preprocessing.…”