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Page 232 Crisafulli et al. Cancer Drug Resist 2019;2:225-41 I http://dx.doi.org/10.20517/cdr.2018.008

use of chromatin immunoprecipitation sequencing in cancer therapy is limited, but it has proven useful in
[72]
stratifying breast cancer subtypes for treatment .

TOP-RANKING NGS TECHNOLOGIES
The birth of NGS sequencing can be placed in 2005, when 454 Life Sciences launched their sequencing-
[73]
by-synthesis tool , as the first instrument of a 2nd generation sequencing technology. Since then, many
companies have developed different NGS products, which are usually based on different, proprietary
procedures and base-detection chemistry. Current best-ranking technologies, comparative performance and
preferential platform applications are presented in Supplementary Tables 2 and 3.

Albeit reference technologies vary widely across NGS platforms, workflows share several steps: (1) DNA
extraction; (2) library preparation, with addition of adaptors and barcodes/indexes; (3) template preparation,
either by bridge amplification or emulsion PCR; (4) automated sequencing [Supplementary Table 3].

The pioneer Roche/454 (www.sequencing.roche.com) sequencing used pyrosequencing, as based on the
detection of pyrophosphate released after nucleotide incorporation in newly synthesized DNA. Currently,
this platform is rarely utilized, because of long run times and high costs.

The Supported Oligonucleotide Ligation and Detection (SOLiD) platform (www.thermofisher.com), is a
short-read sequencing technology based on ligation. DNA fragments are ligated to oligonucleotide adapters,
attached to beads, and clonally amplified by emulsion PCR. Beads with clonally amplified template are
immobilized onto a derivatized-glass flow-cell surface, and sequencing is started by annealing a primer
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oligonucleotide complementary to the adapter . At each cycle the complementary strand is removed and
a new sequencing cycle starts at the position n-1 of the template. Sequencing cycles are repeated until each
base is sequenced twice. The recovered data from the color space can be translated to letters of DNA bases
and the sequence of the DNA fragment is assembled .
[75]
The IonTorrent platform (www.thermofisher.com) is based on sequencing by synthesis and the detection
is based on solid state pH meters, which measure the hydrogen ions that are released during DNA
polymerization.

Illumina (www.emea.illumina.com) currently occupies the most prominent segment of the NGS market. This
technology is based on sequencing by synthesis of template-complementary strands and on fluorescence-
based detection of reversibly blocked terminator nucleotides .
[76]

All technologies above (2nd generation sequencing) require demanding protocols with serial PCR steps
that results in an increased time of processing and cost. Moreover, genome regions with high density of
repeats are difficult to decipher when using short reads . With the entry in the market of 3rd generation
[75]
sequencers, several such hurdles have been overcome. To date, popular platforms are the Pacific Biosciences
and the Oxford Nanopore technologies.

Pacific Biosciences use single molecule real-time (SMRT) technology. Library preparation leads to a
closed circular DNA molecule by ligating an adaptor molecule to both ends of the target DNA molecule
to be sequenced. The circular DNA molecule is then loaded into a cell containing 150,000 zeptolitre wells
(ZMW) [77,78] . Each ZMW contains a DNA polymerase attached to the bottom and the target DNA fragment
for sequencing. During the sequencing reaction, the DNA fragment is labeled by the DNA polymerase with
[75]
fluorescent nucleotides and the corresponding emitted signal is recorded (www.pacb.com) .

The Nanopore technology identifies DNA bases by measuring changes in electric conductivity generated as
DNA strands pass through a biological pore. The chemical differences of each base would result, in theory,