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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Cancer J. 2018 Mar-Apr;24(2):93–103. doi: 10.1097/PPO.0000000000000311

Table 2.

NGS technologies used for liquid biopsy analyses.

NGS Technology
[% mutant detection]		 Description
Ion AmpliSeq™ MiSeq/HiSeq/MiniSeq Illumina [1%–10%] “Sequencing by synthesis” – a complementary strand of DNA is built upon a template strand. Sequencing is based on the detection of hydrogen ion released (Ion) or use of fluorescent readout (Mi/Hi/MiniSeq) during DNA polymerization.36
CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) [~0.02%] Combines library preparation methods for low DNA input masses with a multiphase bioinformatics approach to design a 'selector', consisting of biotinylated DNA oligonucleotides that target recurrently mutated regions. To monitor ctDNA, the selector is applied to primary tumor DNA to identify a patient's cancer-specific genetic aberrations before being applied to ctDNA.35 (Fig. 4A)
Duplex Sequencing (Duplex-Seq) [0.01%] Uses degenerate molecular tags to label each DNA molecule with its own unique DNA sequence. By tagging duplex DNA with adapters containing random but complementary double-stranded nucleotide sequences, it traces every sequence read back to one of the two strands of the original double-stranded DNA molecule. After adapter ligation, the individually labeled strands are PCR-amplified to create sequence families that share the same tag sequences derived from each of the two single parental strands. Following sequencing, members of each tag family are grouped, and a consensus sequence is established for each of the two strands to form “single-strand consensus sequences”. The two complementary consensus sequences derived from the two strands of an individual DNA duplex are then compared with each other, and the base identity at each position is retained only if the two strands match perfectly at that position, yielding a “duplex consensus sequence”. Polymerase errors appear in only one of the two DNA strands and thus are not counted as real mutations.70,71 (Fig. 4B)
Safe-sequencing system (Safe-Seq) [~0.02%] A unique molecular identifier (UMI) approach to detect rare variants via amplification of each tagged template to create UMI families. The abundance of each UMI is used to distinguish between rare mutations and technical errors and can also be used to correct for PCR amplification bias.33 (Fig. 4C)
Molecular barcoding method [0.1%] Relies on semi-degenerate barcoded adapters and personalized panels of biotinylated baits that target somatic mutations previously identified via the sequencing of tumor/liquid biopsies.72 (Fig. 4D)
Tagged amplicon deep sequencing method (TAm-Seq™) [0.25%–0.5%] Combines library preparation and statistically-based analysis algorithms to identify and quantify cancer mutations.34 (Fig. 4E)
Proximity Sequencing (Pro-Seq) [0.003%] Involves linking multiple copies of a single DNA template at the 5’ end early in the workflow so that the sequences of all molecules in a linked complex are nominally the same (except for any errors made in their derivation from the parent strand). The linking is arranged that both senses of the starting template can be represented in a single linked complex, providing duplex information. The linked complex is sequenced directly so that the multiple linked copies seed a single sequencing cluster/colony. A single cluster represents the aggregation of multiple redundant members of a family instead of a single molecule. Linked dsDNA fragments populate one cluster on an Illumina flow cell. dsDNA sequencing errors are compensated for by comparing the two strands of the DNA molecule.73 (Fig. 4F)
CypherSeq [0.001%–0.01%] Two biotinylated, target-specific primers are used for each DNA strand. DNA is ligated into circular bacterial vectors that contain the targeted region of interest, and each strand is barcoded with a short sequence of nucleotides and then amplified by rolling-circle amplification (RCA). RCA product is a biotinylated strand concatenated with many copies of the template. Errors introduced during amplification are along the concatenated strand and are eliminated computationally. Concatenate is biotinylated; therefore, the strand can be pulled and template kept.74 (Fig. 4G)