Single-molecule sequencing technology, a novel method for gene sequencing, utilizes nano-sized materials to detect electrical and fluorescent signals. Compared to traditional Sanger sequencing and next-generation sequencing technologies, it offers significant advantages, including ultra-long read lengths, rapid sequencing, and the absence of amplification steps, making it widely applicable across various fields. By examining the development and components of single-molecule sequencing technology, it becomes clear that its unique characteristics provide new opportunities for advancing metrological traceability. Notably, its direct detection capabilities offer a novel approach to nucleic acid metrology. This paper provides a detailed overview of library construction, signal generation and detection, and data analysis methods in single-molecule sequencing and discusses its implications for nucleic acid metrology.

The exploration of deep-sea microorganisms is transitioning from ex situ laboratory analysis to in situ real-time monitoring. While in situ technologies offer unprecedented access to microbial activities in their natural extreme habitats, they face a critical, yet often overlooked, bottleneck: the absence of a robust metrological framework. This lack of standardized calibration, traceability, and reference materials results in data that are often irreproducible, device-specific, and incomparable across studies, severely undermining scientific discovery and resource assessment. This review provides a systematic analysis of the current landscape of deep-sea microbial detection technologies, categorizing them by their operational principles and critically evaluating their performance, limitations, and metrological readiness. By synthesizing the technological challenges with the principles of metrology, we identify the fundamental gap between advanced sensing capabilities and the lack of in situ measurement standards. To bridge this gap, we propose an innovative “laboratory simulation–in situ detection–remote calibration” trinity calibration system. This framework establishes a complete metrological traceability chain tailored for extreme deep-sea conditions, aiming to transform isolated sensor data into globally comparable, scientifically robust, and industrially actionable information, thereby paving the way for precision deep-sea biology and governance.