Sequencing

Illumina sequencing 

Illumina’s innovative and flexible sequencing system enables a broad array of applications in genomics, transcriptomics, and epigenomics. Libraries are prepared from genomic DNA or RNA, then immobilized on the surface of a flow cell designed to present the DNA in a manner that facilitates access to enzymes while ensuring high stability of surface-bound template and low non-specific binding of fluorescently labeled nucleotides. Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity with total densities on the order of more than one million single-molecule clusters per square millimeter.

Sequencing by Synthesis

Sequencing by synthesis (SBS) technology uses four fluorescently labeled nucleotides to simultaneously sequence the tens of millions of clusters on the flow cell surface.  During each sequencing cycle, a single labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain. The nucleotide label serves as a terminator for polymerization.  After each dNTP incorporation, the fluorescent dye is imaged to identify the base and later enzymatically cleaved to allow incorporation of the next nucleotide. Since all four reversible terminator-bound dNTPs (A, C, T, G) are present as single separate molecules, natural competition minimizes incorporation bias. Base calls are made directly from signal intensity measurements during each cycle, thereby reducing raw error rates. The end result is highly accurate base-by-base sequencing that eliminates sequence-context specific errors, enabling robust base calling across the genome.

Oxford Nanopore sequencing 

The Oxford Nanopore sequencing technology is part of the fourth-generation DNA sequencing technology which offers advantages in all areas of research, such as microbiology, environmental research, microbiome, human genetics, cancer research, plant and animal research and more. The MinION device is the only portable real-time device for DNA and RNA sequencing. Nanopore sequencing offers, for the first time, direct RNA sequencing, as well as PCR-free cDNA sequencing.

Ultra-long read lengths are possible (hundreds of kb) as you can choose your fragment length. The MinION streams data in real time so that analysis can be performed during the experiment and workflows are fully versatile.

A range of preparation kits are available to support analysis of DNA, cDNA and direct RNA. Data is streamed in real time, allowing running of the instrument until sufficient data is generated to answer the researcher’s question.

How does it work

Nanopore sequencing works by driving DNA or RNA molecules through thousands of tiny tubes, called nanopores, which sit in a membrane. As the molecules move through a nanopore, each base creates a disruption in the electric current. These disruptions are analyzed to determine the sequence. This technology allows researchers to watch DNA and RNA being sequenced in real-time, and even interact with their experiments while they are running. As nanopore sequencing looks at largely unprocessed DNA, it can pick up important epigenetic changes, such as whether a gene is turned on or off. Similarly, since DNA is not broken into small sections for sequencing, it also detects large structural changes.