Massive parallel sequencing

These technologies use miniaturized and parallelized platforms for sequencing of 1 million to 43 billion short reads (50 to 400 bases each) per instrument run.

Third, the spatially segregated, amplified DNA templates are sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step.

For imaging systems which cannot detect single fluorescence events, amplification of DNA templates is required.

In the aqueous water-oil emulsion, each of the droplets capturing one bead is a PCR microreactor that produces amplified copies of the single DNA template.

The flow cell is exposed to reagents for polymerase-based extension, and priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface.

Repeated denaturation and extension results in localized amplification of DNA fragments in millions of separate locations across the flow cell surface.

[24][25] This technology was filed for a patent in 1997 from Glaxo-Welcome's Geneva Biomedical Research Institute (GBRI), by Pascal Mayer, Eric Kawashima, and Laurent Farinelli,[4][5] and was publicly presented for the first time in 1998.

AT-rich and GC-rich target sequences often show amplification bias, which results in their underrepresentation in genome alignments and assemblies.

In either approach, DNA polymerase can bind to the immobilized primed template configuration to initiate the NGS reaction.

This principle of sequencing-by-synthesis has been used for almost all massive parallel sequencing instruments, including 454, PacBio, IonTorrent, Illumina and MGI.

The principle of Pyrosequencing was first described in 1993 [1] by combining a solid support with an engineered DNA polymerase lacking 3´to 5´exonuclease activity (proof-reading) and luminescence real-time detection using the firefly luciferase.

This approach uses reversible terminator-bound dNTPs in a cyclic method that comprises nucleotide incorporation, fluorescence imaging and cleavage.

In its simplest form, a fluorescently labelled probe hybridizes to its complementary sequence adjacent to the primed template.

The method of real-time sequencing involves imaging the continuous incorporation of dye-labelled nucleotides during DNA synthesis: single DNA polymerase molecules are attached to the bottom surface of individual zero-mode waveguide detectors (Zmw detectors) that can obtain sequence information while phospholinked nucleotides are being incorporated into the growing primer strand.

Pacific Biosciences uses a unique DNA polymerase which better incorporates phospholinked nucleotides and enables the resequencing of closed circular templates.

[37][38] In 2015, Pacific Biosciences released a new sequencing instrument called the Sequel System, which increases capacity approximately 6.5-fold.