Comparative Frameworks for Next- and Third-Generation Sequencing in Genomic Applications

Abstract

Next-generation sequencing transformed genomics by shifting from capillary separations to massively parallel molecular imaging, while third-generation platforms extended read lengths and enabled direct sensing of native nucleic acids. Against this historical backdrop, technical choices now revolve around chemistry–physics trade-offs, error models, and computational inference rather than a single dominant instrument class. This paper develops a comparative, methods-focused analysis of the dominant architectures, including sequencing-by-synthesis with cyclic fluorescent interrogation and single-molecule modalities that detect polymerase-mediated incorporations or ionic-current perturbations through nanometer pores. Assay steps from library construction to basecalling are treated as a coupled stochastic pipeline whose performance hinges on fragment length distributions, molecular tagging, transduction bandwidth, and priors embedded in learning-based decoders. A unified modeling view is proposed for coverage, assembly continuity, and haplotype resolution across variant scales, relating platform-specific error spectra to algorithmic robustness in consensus, phasing, and structural discovery. Special attention is given to epigenetic and transcriptomic readouts, where native modification detection and full-length isoform capture yield qualitatively new observables that cannot be retrofitted from short fragments alone. Economic and operational considerations are formalized through throughput and cost functions that incorporate flowcell physics, pore occupancy, polymerase kinetics, and sample complexity. By mapping the tensions between accuracy and contiguity, speed and depth, and standardized pipelines and bespoke analyses, the paper articulates design implications for population sequencing, clinical validation, and multi-omic integration. The resulting framework clarifies when short-read depth remains optimal, when long-read continuity is decisive, and how hybrid and adaptive strategies best exploit the strengths of both generations.