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in esophageal adenocarcinoma making this technology a possibil-
ity for clinical use and provides patient-specific information.
In conclusion, among the three approaches of NGS, whole-
genome sequencing is the most comprehensive but expensive,
whereas whole-exome sequencing is cost-effective but it only
works for the known genes. Thus, second-generation sequencing
methods can provide a complete picture of the genome of esopha-
geal adenocarcinoma by detecting and discovering different types
of alterations in esophageal adenocarcinoma that would help in
diagnostics and will further help in developing personalized medi-
cine in esophageal cancer.
Many instruments could perform next-generation sequencing
based on different chemistries. For example, sequencing by synthe-
sis uses specially designed fluorescent-labeled terminator nucleo-
tides, which allow the chain termination process to be reversed.
The terminator contains a fluorescent label, which can be detected
by a camera generating a temporal series of color images that can
be computationally converted into sequence reads [ 18 ]. Another
technology uses ligation of fluorescently labeled hybridization
probes to determine the sequence of a template DNA strand, two
bases at a time as a program for sequencing [ 1 ]. Alternative method
of sequencing is pyrosequencing, which depends on the detection
of pyrophosphate released during nucleotide incorporation [ 19 ].
In the following discussion, we would present the use of one
of the benchtop sequencers based on semiconductor technology.
In this technology, a nucleotide is incorporated into the DNA mol-
ecules by the polymerase; a proton is released resulting in the
change in pH helping the sequencer to recognize whether a nucle-
otide is added or not [ 20 ].2 Materials
Nucleic acid from various sources—including formalin fixed paraf-
fin embedded (FFPE) tissue, and cell-free DNA—can be used as
the starting material.
Custom Panels.
The Ion AmpliSeq™ Library Kit 2.0 provides reagents for pre-
paring eight libraries.
Should be stored at −30 to − 10 °C. It consists of the following
reagents:
5× Ion AmpliSeq™ HiFi Mix.
FuPa Reagent.
Switch Solution.
DNA Ligase.Suja Pillai et al.