SMRT

SMRT sequencing, or single-molecule real-time sequencing, is a DNA sequencing technology developed by Pacific Biosciences (PacBio). It enables observation of DNA synthesis by a DNA polymerase in real time, using fluorescently labeled nucleotides and zero-mode waveguides (ZMWs) to detect incorporation events on individual DNA molecules. Sequencing libraries consist of SMRTbell templates—circular DNA constructs that allow the polymerase to read the template multiple times, increasing accuracy.

The technology produces long reads, with typical read lengths ranging from 10 to 25 kilobases, and, with circular consensus sequencing (CCS), can generate highly accurate reads (HiFi reads) with accuracies above 99.9% albeit shorter read lengths. Raw reads have higher per-base error rates, which are mitigated by consensus approaches.

Applications include de novo genome assembly, resolving repetitive and structural variation, full-length transcript sequencing, and complex regions difficult for short-read technologies. SMRT sequencing has been used in human genome projects, plant and microbial genomics, and metagenomics.

Limitations include higher per-base cost, more demanding DNA quality and input, and lower throughput per run compared to some short-read platforms, though newer instruments like Sequel IIe and Revio have expanded throughput and reduced costs per gigabase. PacBio workflows also support SMRTbell libraries and long-range phasing.

In summary, SMRT sequencing provides long, contiguous reads and real-time observation of DNA synthesis, enabling improved genome assemblies and various genomic analyses, at the trade-off of higher DNA input requirements and cost.