Sequencing Technologies
One-Sentence Definition
Sequencing technologies are the instrument platforms that convert DNA/RNA molecules into readable base sequences, differing mainly in read length, per-base accuracy, throughput, and cost.
Simple Explanation
Short-read machines give very accurate small pieces; long-read machines give long pieces that span repeats. Microbial genomics often wants both.
Detailed Scientific Explanation
| Platform class | Read length | Accuracy | Strength | Weakness |
|---|---|---|---|---|
| Illumina (SBS, short-read) | ~100–300 bp | Very high per base | Cheap, high depth, SNP calling | Repeats/plasmids unresolved |
| Oxford Nanopore (ONT) | kb–Mb | Improved but lower raw | Portable, real-time, long | Homopolymer/systematic errors |
| PacBio HiFi | ~10–25 kb | High (consensus) | Complete genomes | Cost, instrument access |
| Sanger | ~700–900 bp | Gold for single amplicons | Confirmation, 16S | Not genome-scale |
Hybrid assembly (Illumina + ONT/PacBio) is the classic route to closed bacterial chromosomes and complete plasmids.
Mechanism
- Illumina: bridge amplification + reversible terminators, imaged cycle by cycle
- ONT: current change as DNA passes through a protein nanopore → basecalling model (itself a neural network)
- PacBio: circular consensus of repeated passes over the same molecule
Clinical Importance
- Turnaround and portability matter for outbreak response (ONT in field settings)
- Complete plasmid reconstruction changes AMR epidemiology conclusions
Research Importance
- Direct RNA sequencing (ONT), methylation detection, structural variants
Diagnostic Relevance
- Underpins Whole-Genome Sequencing and Metagenomics workflows
AMR Relevance
- Short reads may detect a carbapenemase gene but not its Plasmid context — long reads resolve mobility
Related MOCs
Active Recall Questions
- Why does plasmid epidemiology often need long reads?
- Which platform is most portable for field outbreak work?
- What is hybrid assembly?