Next Generation Sequencing (NGS)

What is Next Generation Sequencing (NGS)?

Next Generation Sequencing (NGS) is a technology in which nucleotides are added in parallel to the copying of a DNA strand. When nucleotides are incorporated into the growing DNA strand, a signal is generated that corresponds to the nucleotide type and position. Regardless of the distinct method used to capture nucleotide information, NGS provides a wide snapshot of the genome of interest with higher sensitivity and higher capacity with sample multiplexing.

There are several distinct methods used to generate and capture signals that translate into a DNA sequence.

Nanopore/ONT (Oxford Nanopore Technologies) Sequencing:

• Signal: Change in electrical current when a nucleotide is passed through a nanopore
• Method: The DNA sequence is determined by the change in electrical current

Illumina Sequencing:

• Signal: Nucleotides are labeled with fluorescence and added to the growing DNA strand
• Method: A camera captures an image of the fluorescence signal associated with the identity of each nucleotide; the order of each fluorescence signal translates to the sequence of DNA

Ion Torrent Sequencing:

• Signal: Hydrogen ions are released when a nucleotide is added, which leads to a change in pH
• Method: The sequence of DNA is determined by the changes in pH

PacBio (Pacific Biosciences) Sequencing:

• Signal: Also referred to as Single-Molecule Real-Time (SMRT®) sequencing, fluorescently labeled nucleotides are observed in real-time as DNA is polymerized
• Method: The sequence of DNA is determined by the timing and order of the incorporated fluorescently labeled nucleotides

What is the importance of next generation sequencing?

NGS is important for any molecular biologist because of its speed. With the ability to sequence millions of DNA strands simultaneously in a single run, the fast turnaround time allows for high sample volumes and the ability to sequence up to thousands of genes or gene regions. NGS technology enables the scientist to expand their DNA snapshot – gaining a wider perspective and understanding with comprehensive genomic coverage of their sample.

What is the difference between NGS and Sanger sequencing?

NGS sequences millions to billions of DNA fragments in parallel, allowing for significantly high-throughput and rapid, cost-effective generation of genomic data. Sanger sequencing traditionally produces longer read lengths than NGS, at lower throughput since DNA fragments are read one at a time. Sanger sequencing is advantageous for certain applications, such as validating DNA clones, sequencing long stretches of DNA, and resolving complex genomic regions.