Third-Generation Sequencing

The next era of DNA sequencing technology, termed third-generation sequencing (TGS), has gained a place in biology as a way to study genomes, transcriptomes, and metagenomes at an unprecedented resolution. These technologies work by single-molecule sequencing and provide:

  • Long reads with no amplification.
  • Direct detection of epigenetic modifications on native DNA.
  • Direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms.
  • Uniform coverage of the genome as they are not as sensitive to GC content as short-read platforms.

The UMGC offers long-read sequencing solutions from PacBio and Oxford Nanopore, giving researchers a wide range of cutting-edge sequencing solutions to fit any project scale, budget, and turnaround time. As a PacBio Certified Service Provider with five years of experience with the Sequel II system, we undergo standardized PacBio certification to demonstrate that we can generate high quality data using the most up-to-date workflows.

TGS Single-molecule Technologies

There are two TGS technologies currently available at the UMGC: 1) single-molecule real-time (SMRT) sequencing on the PacBio Sequel II, and 2) nanopore sequencing on the Oxford Nanopore Technologies (ONT) GridION X5.

The Sequel II and GridION X5 have varying throughput, but both work by reading the nucleotide sequences at the single-molecule level and produce substantially longer reads (10 kb-100 kb) than current NGS methods. Raw reads do have an error rate higher than Illumina systems; however, these errors are random and so error correction algorithms can improve the per-nucleotide accuracy to >99.99% or more with sufficient coverage.

Though both are single-molecule sequencing technologies, the technology behind the PacBio and ONT platforms are considerably different:

  • The most established technology, the SMRT technology by PacBio, uses eight million zero-mode waveguides (ZMW) - microwells bound with a DNA polymerase - to capture signal from the incorporation of fluorescent-labeled nucleotides during DNA replication of SMRTbell templates. The detection system records a movie of light pulses that are converted to base calls to create a continuous long read from each ZMW. 
  • The most recently released TGS technology is ONT’s and uses nanopores embedded in an electro-resistant membrane.  Each nanopore corresponds to its own electrode connected to a channel and sensor chip, which measures the electric current that flows through the nanopore. When a molecule passes through a nanopore, the current is disrupted to produce a characteristic signal. This signal is then decoded using basecalling algorithms to determine the DNA or RNA sequence in real-time.

Questions?

Whether a researcher is new to sequencing or one of our regular users, we can help determine which instrument and application best meet a project’s goal. Researchers are encouraged to contact our NGS team for a consult on experimental design, library creation methods, or sequencing options at next-gen@umn.edu.