Episode notes
Single-molecule real-time (SMRT) sequencing, pioneered by Pacific Biosciences and complemented by Nanopore sequencing technologies, represents a major advance over short-read sequencing approaches. The key breakthrough was the elimination of the DNA amplification step, allowing individual DNA molecules to be sequenced directly and avoiding biases introduced during PCR amplification. This direct sequencing approach also enabled the generation of much longer reads, typically in the range of 10–20 kb and beyond, providing valuable long-range information for genome assembly, structural variant detection, and resolving repetitive regions.
However, early single-molecule sequencing technologies were associated with substantially higher error rates, approaching 10–15% compared with the approximately 99.9% base-level accuracy of Illumina short reads. These errors were largely random and could be eliminated by sequencing the same molecule multiple times and alimenting them by improved consensus algorithms. Recent advances, particularly high-fidelity (HiFi) sequencing from PacBio, have dramatically improved accuracy while retaining long-read capabilities, bridging the gap between the accuracy of short reads and the genomic resolution offered by long reads.