HiFi sequencing does not require PCR amplification because it is a single-molecule, real-time (SMRT) sequencing technology. Instead of copying DNA millions of times before sequencing, HiFi reads are generated by observing a single native DNA molecule as a DNA polymerase synthesizes it in real time.
Each DNA fragment is converted into a circular SMRTbell template. During sequencing, the polymerase makes multiple passes around the same molecule. These repeated observations are combined through Circular Consensus Sequencing (CCS) to produce a highly accurate HiFi read (Q30+), without the need for amplification.
Avoiding PCR provides several advantages:
- Reduced amplification bias, including improved representation of GC-rich or complex regions
- More uniform genome coverage
- Preservation of native DNA characteristics
- Direct detection of base modifications
By achieving accuracy through consensus of multiple passes—rather than amplification—HiFi sequencing delivers long, highly accurate reads while maintaining the integrity of the original DNA molecule.