The adapter ligation efficiency and the number of PCR cycles are
two critical factors for this procedure. For hybridization capture,
first, one should design a panel consistent with the particular appli-
cation and then order the probes to a manufacturer; especially
NimbleGen SeqCap EZ could be a good choice. The Illumina
sequencing platform is usually applied in the last sequencing step.
Figure2 demonstrates how blood samples are processed and how
the libraries are prepared and sequenced.1.3 Difficulties of
Analyzing ctDNA
NGS Data
Cell-free tumor DNA is only a small fraction of cell-free DNA,
especially for samples from early-stage cancer patients. This fact
makes it hard to detect tumor-specific mutations. Furthermore,
PCR and sequencing errors, DNA oxidative damages, and
software-introduced artifacts can produce a high level of noise
and introduce many false-positive mutations.
The amount of tumor-specific DNA can vary greatly from less
than 0.01% to more than 90% [3]. The variability of ctDNA abun-
dance is associated with tumor burden, stage, vascularity, cellular
turnover, and response to therapy. Theoretically DNA alteration of
any fraction is detectable via deep sequencing with sufficient num-
ber of molecules. However, amplification bias during PCR ofFig. 1Applications of ctDNA for cancer diagnosis and therapy. Cell-free DNA can be evaluated by different
techniques like digital PCR (dPCR) or next-generation sequencing (NGS). Digital PCR can only cover a few
target loci, while NGS can cover millions of loci, including the whole exome or even whole genome. CtDNA
testing can be applied to search for targeted therapy drugs, evaluate treatment response, and detect early-
stage cancers
Bioinformatics Analysis for Cell-Free Tumor DNA Sequencing Data 69