Pacific Biosciences Sequencing
The new Pacific Biosciences Sequel system builds upon their Single-Molecule, Real-Time (SMRT) technology and delivers higher throughput, increased scalability and lower sequencing costs compared to the PacBio RS II.
The Sequel system utilizes the DNA sample as a direct template for the sequencing reaction in order to sequence individual DNA molecules in real time. Ligation of hairpin adapters converts the double-stranded DNA molecules into a circular template that enables continuous sequencing of both the forward and reverse strands without the need for amplification. The unique circular structure allows for continuous long reads of large-insert libraries or circular consensus sequencing of smaller insert sizes. Extra-long read lengths and high consensus accuracy make the Sequel a helpful tool for finding SNPs and sequencing through GC-rich areas of the genome.
Strengths and Applications
- Rapid, cost-effective generation of high-quality, de novo whole genome assembly
- No PCR bias, uniform coverage for targeted sequencing
- Direct base modification detection for epigenetic studies
- Long read lengths
- Single-molecule resolution of complex populations
- High consensus accuracy
Sequencing on the Sequel
The basic sequencing "unit" for the Sequel is the SMRTcell, analogous to a flowcell in Illumina sequencing. SMRTcells do not have lanes, unlike flowcells, so each sample is sequenced on its own SMRTcell. There are no "read lengths" like in Illumina sequencing; instead, you choose a length of time to continuously run the sequencing reaction, known as a "movie time". The maximum movie time for the Sequel is 10 hours.
There are two different methods of loading the SMRTcell: diffusion loading and Magbead loading. Fragment size largely determines which loading strategy you will use. For smaller fragment sizes (<7.5kb), diffusion loading is generally the recommended method. In diffusion loading, SMRTbell library molecules with bound polymerase are immobilized and diffuse to the bottom of the ZMW wells after removal of excess polymerase and primer with cleanup beads. For fragments >7.5kb, Magbead loading is recommended, which removes adapter dimers and short insert sizes to maximize read lengths.