Next-Gen Sequencing Is A Numbers Game | August 18, 2014 Issue – Vol. 92 Issue 33 | Chemical & Engineering News

NextGen #Sequencing Is A Numbers Game Overview of contenders w/ snippets categorizing #chemistry, eg seq-by-synthesis


So far, Illumina leads the race. In January, the San Diego-based firm

launched its HiSeq X Ten system with a price tag of $10 million.
Consisting of 10 ultra-high-throughput sequencers, each capable of
generating up to 1.8 terabases of data in less than three days

Illumina uses a sequencing-by-synthesis method. After DNA fragments

are amplified on a chip, sequencing occurs by synthesizing a DNA
strand complementary to the target strand by enzymatically attaching
fluorescently labeled nucleotides one at a time. When reactions occur,
the labels are optically imaged to identify what was attached, and the

cycle is repeated.

Thermo Fisher holds second place in the NGS market, with about 16% of
sales. ….ABI launched its first NGS system based on sequencing by
oligonucleotide ligation and detection, known as SOLiD.

Unlike highly accurate but less parallelizable Sanger methods, NGS

systems carry out massive numbers of reactions, or sequence reads, at
one time. Like Illumina’s approach, SOLiD uses sequencing by synthesis
of amplified DNA fragments on either a bead or chip. Instead of
nucleotides, it uses fluorescently labeled probes that are repeatedly

ligated to the growing strand, optically imaged, and cleaved off. How
long these processes can be kept going determines the “read length”
that can be sequenced in a run.

The first lower-cost, nonoptical system appeared in 2010 after Life

Technologies—now part of Thermo Fisher and formed from the 2008 merger
of ABI and Invitrogen—acquired Ion Torrent for $725 million. Its
systems use sequencing by synthesis, but with unlabeled nucleotides on
a semiconductor chip. The chip electrically senses the release of

hydrogen ions when bases attach. The full sequence is read by
sequentially adding bases and tracking reactions across millions of

Pacific Biosciences’ single-molecule real-time sequencing is a
sequencing-by-synthesis approach that doesn’t use an amplified set of
DNA fragments and doesn’t require stopping and starting the reaction

to add reagents and image results. Reactions on individual DNA
molecules are tracked in real time across 150,000 nanoscale wells
where isolated polymerases read the DNA and incorporate fluorescently
tagged nucleotides. Because detection occurs only at the bottom of the

wells, the background noise from the other reactions is reduced.

Stability of the sequencing process depends in large part on the
polymerase. Pacific Biosciences has modified a simple bacteriophage
enzyme, slowing it down so that it incorporates about three bases per

second and its detector can keep up.


Most interest has been in the U.K.’s Oxford Nanopore Technologies as
it moves closer to launching a new sequencing device. Its MinION uses

protein nanopores held in a polymer membrane to sequence
single-stranded DNA in real time. Individual bases are identified
through changes in electrical current as a linear, single-stranded DNA
molecule moves through a nanopore.


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