Table 1 Comparison of ctDNA mutation analysis technologies [159]

First generation and next generation sequencing

Single locus or limited multiplex assays

PCR-based:

· Droplet Digital PCR (ddPCR) [140]

· BEAMING [31]

· Intplex [149]

· Detection and quantification of selected loci, cancer hotspot mutations and small number of mutations

· Widely used

· High sensitivity

· Sampling error can happen when samples need to split into multiple reactions

· Very low copy numbers of mutant DNA impairs the overall performance

· Only detects previously known mutations

Enrichment for mutant alleles:

· COLD-PCR [82]

· SCODA [95]

· NaME-PRO [136]

Next generation sequencing

Targeted sequencing assays

Amplicon-based method:

· SiMSen-Seq [144]

· TAM-Seq [38]

· Enhanced TAM-Seq [41]

· Safe-SeqS [71]

·AmpliSeq [143]

·MiSeq [69]

·NextSeq [67]

·HaloPlex [83]

· Genotyping

· Interrogating numerous mutations (SNP, CNV, insertion, deletion, structural variation)

·Individual exons of interest to the whole exome

· Reduced background error rates because of higher coverage compared to WGS

· Detection of allele fractions below 0.1%

· Even with limited amounts of input samples, sensitivity can be further enhanced

· Only detecting mutations in targeted genes/locations

· Limited detection of fusions

Hybridization capture:

· CAPP-Seq [104]

· TARDIS [97]

· SureSelect [74]

· SeqCap [26]

· HiSeq [81]

Non-targeted assays or Genome-wide

Whole genome sequencing (WGS):

· Plasma-Seq [58]

· PARE [75]

· Identification of amplifications and deletions

· Detection of fetal aneuploidies

· Investigation of genomic alterations without previously knowledge

· Characterization of the whole molecular landscape

· Limited detection in low tumour purity samples (< 25%) allele fractions sensitivity poor below low coverage

· Limited sensitivity for profiling early-stage cancer

· High cost

Amplicon-based:

· FAST-SeqS [70]

· mFast-SeqS [9]

Third generation sequencing

Targeted and whole genome sequencing

· Oxford Nanopore Technology (ONT)

· CyclomicsSeq [92]

·Detection of a wide range of variants from SNV to structural variants

· Methylation of cfDNA

· Rapid turnaround time

· Flexible accessibility

· Cost-effective

· Unrestricted read length (20 bp to 4 Mb)

· Relatively high error rates