from other tissues. Since both normal and tumor cells shed DNA
into the circulation, in general there is a relatively low abundance of
tumor-specific cfDNA in the total cfDNA. Before a DNA fragment
is sequenced, we do not know whether it is derived from a tumor
cell. So we can only do cfDNA sequencing in all at this stage and
have no way to merely do ctDNA sequencing. However, for appli-
cations in cancer field, ctDNA is a term much better known and
more widely used by researchers. So in this chapter, we will use both
ctDNA sequencing and cfDNA sequencing. When we mention
ctDNA sequencing for cancer patients, be noted that it is the
same as cfDNA sequencing.
In general, cfDNA dynamically reflects the overall state of the
body. The tumor-specific ctDNA are good biomarkers for early
detection and prevention, assessing minimal residual disease and
prognosis, monitoring tumor burden, and guiding therapies. It
allows a relatively noninvasive repeated serial sampling for continu-
ous monitoring of disease. Moreover, the ability to identify specific
drug-sensitizing or resistance mutations in the blood of cancer
patients makes liquid biopsy a good alternative method for tissue
biopsies, which is often difficult or impossible to obtain for late-
stage cancer patients using the conventional methods. Although
lots of studies support these concepts, the use of cfDNA in clinical
practice is still in its infancy and requires rigorous prospective
validation studies to demonstrate the benefit of this promising
analyte to facilitate the clinical decision making. Figure1 illustrates
how ctDNA can be applied for cancer diagnosis and therapy.1.2 How Is ctDNA
Sequenced
As there is a relatively low abundance of ctDNA in the total cfDNA,
highly sensitive techniques are required for ctDNA detection.
Advancements in next-generation sequencing (NGS) have made it
possible to detect low occurrence mutations in a heterogeneous
population [5].
The main steps for ctDNA sequencing include sample proces-
sing and cfDNA extraction, NGS library construction, hybrid cap-
ture, and sequencing.
CtDNA can be extracted from bodily fluids like plasma, cere-
bral spinal fluid, urine, pleural effusion, etc. The choice of material
is related to the clinical or research purpose. The amount of cfDNA
extracted from 1 ml plasma is relatively low, and an accurate quan-
tification of cfDNA is necessary for the subsequent library con-
struction procedure. A classical library construction process on
Illumina platform includes end repair to produce blunt-ended
and 5^0 -phosphorylated dsDNA fragments; A-tailing, during which
dAMP is added to the 3^0 -ends of blunt-ended dsDNA library frag-
ments; adapter ligation, during which dsDNA adapters with
30 -dTMP overhangs are ligated to 3^0 -A-tailed library fragments;
and library amplification, which employs PCR to amplify library
fragments carrying appropriate adapter sequences on both ends.68 Shifu Chen et al.