Molecular methods for colorectal cancer screening: Progress with next-generation sequencing evolution

Table 1 Summary of the main diagnostic approaches for colorectal cancer screening

Technology	Approach	Sample types	Targeted and colorectal marker	Sensitivity/specificity for CRC	Advantages	Disadvantages
Chemical and immunochromatographic test	FOBT	Stool	Heme of hemoglobin	4%-25%/95%	(1) Non-invasive; (2) Reduction of mortality (asymptomatic patients); (3) Colorimetric indicator; (4) Rapid and easy-to-carry out (self-testing); and (5) Commercially available test	(1) Low sensitivity for non-bleeding adenoma and advanced adenoma; (2) Specificity influenced by diet or drugs; (3) Must be done annually; (4) Risk of false positive results; (5) Three consecutive samples needed; (6) Only detects the blood present in the external layer of the stool; and (7) Confusing interpretation of the test results
	FIT	Stool	Globin molecules	62.0%-100%/94.9%	(1) Easy to use; (2) Flexible cutoff concentration; (3) Sensitive to low concentrations of globin; (4) Single sample needed; (5) Combined with FOBT inferred mortality; and (6) No dietary restriction	(1) Insensitive to digested hemoglobin; (2) Poor sensitivity for advanced adenoma; (3) Sensitivity based on threshold value of hemoglobin; and (4) Detect more distal neoplasms
Visual inspection	FS	Distal colon	Polyps	100%/100%	(1) Reduce colorectal cancer mortality and incidence; and (2) High susceptibility to detect adenomas	(1) Invasive process; (2) Not suitable for diabetic or psychotropic patients; (3) Expensive; (4) Serious harms for colonoscopy that increase with age; (5) Sigmoidoscopy was not effective for female screening (high risk for proximal colorectal cancer); and (6) Moderate-to-severe pain was reported for patients (bleeding, anxiety, etc)
	TC	Entire colon
Sanger sequencing methodology	Single gene sequencing	Tissue; liquid biopsy	A specific gene in human tumor DNA cells	High sensitivity (input of DNA mutated quantity < 1%)	(1) Non-invasive (blood/liquid biopsy); (2) Some mutations were prominent in colorectal cancer; (3) Bioinformatic analysis not required; (4) Simple and less time consuming; and (5) No specialized instrument in laboratory	(1) Requires high-quality DNA; (2) Heterogenous mutations genes; (3) Risk of contamination with normal tissue; and (4) Low coverage sequencing
	ddPCR	Liquid biopsy. Tissue	Short amplicon sizes (< 100 bp) of human DNA	Very high sensitivity (input of mutated DNA quantity < 0.1% even with degraded DNA)	(1) Monitoring tumor burden in response to treatment and indicator of disease progression; (2) Precise measurement of copy number of mutated DNA and lower probability error (without standard samples); (3) Minimally invasive process; (4) Detects specific mutations; (5) Independent prognostic factor; and (6) Large target mutation	(1) No ability to detect benign lesions from plasma due to insufficient tumor burden; (2) Need an expensive instrument; (3) Limited prime-probe sets for each single nucleotide change; (4) No information in tumor-associated protein profiling; (5) Possibility of contamination with normal tissue; (6) Not strictly tumor specific; and (7) Necessity of cell search system
	MT-sDNA	Stool	Specific genes in human tumor DNA cells	66%-94%/90%-96%	(1) Non-invasive test; (2) Acceptable cost; (3) Potential credibility; (4) No dietary restrictions (including food and medications); and (5) Widespread accessibility and multiple commercialized prototypes	(1) Lack of standardization or optimization of fecal DNA panels for high sensitivity and specificity; (2) Risk of contamination by microbial DNA; (3) No defined optimal interval for screening individuals; (4) Poor sensitivity for advanced adenoma; and (5) Must be repeated every 3 years
	Idylla system	Tissue; liquid biopsy	Specific genes in human tumor DNA cells	High sensitivity (input of DNA mutated quantity < 1%)	(1) Fully automated; (2) Real-time based-PCR molecular diagnosis system; (3) Without pre-analytical DNA extraction; (4) Lower cost and time requested for results; (5) Easily implemented in routine laboratory workflow; (6) Wide range of CRC-related mutations; and (7) Very sensitive to detect the most common CRC mutation	(1) No detection of complex genomic variants; (2) Unknown mutations were not detected; (3) Cannot detect rare and complex genomic variants not included in the reference range; and (4) Less suitable when new gene mutations appear
	Custom panel sequencing	Tissue; liquid biopsy	Specific genes in human tumor DNA cells	95%-100%/99%-100%	(1) Decreased sequence cost; (2) Greater sequencing depth; (3) Simple and less time consuming; (4) Robust and tissue efficient; (5) Massive parallel multigene sequencing; and (6) Provide additional information (TMB levels/relevant mutated genes/heredity cancer genes)	(1) Low coverage sequencing; (2) There is no standardized procedure; and (3) Relatively long turnaround time of 3 d
Next generation sequencing	WGS/WES	Tissue; liquid biopsy	All exome and all genome in human tumor DNA cells	95%-100%/99%-100%	(1) Detection of large-scale mutations; (2) High coverage sequencing; (3) Complete definition of the genomic landscape for WGS; and (4) Complete mutation analysis panel without the repeated testing cost and reuse of material	(1) Require bioinformatics specialists; (2) Expensive; (3) Require good quality DNA; and (4) Relatively long turnaround time of 3 d
	Third generation sequencing	Tissue/stool/liquid biopsy	(1) Specific genes/WES/WGS in human tumor DNA cells; and (2) Microbes in stool	95%-100%/99%-100%	(1) Identification of large-scale rearrangement; (2) Sequencing errors do not release rearrangement; (3) High coverage sequencing; and (4) Fast and real time molecular diagnosis system	(1) High percentage of somatic errors; (2) Require bioinformatic specialists for assembling and analysis in laboratory; (3) Need specialized equipment in laboratory; and (4) Cannot detect some somatic mutations
	Metagenomic analysis	Stool/tissue	Microbial DNA in stool by Shotgun (all the DNA) or metabarcoding DNA (16S, ITS1, ITS2, 18S, etc)		(1) Microbial flora was more abundant than human cells in stool; (2) Benign lesions do not release human cells in stool; (3) Noninvasive diagnostic test; (4) Microbiota seems to play a role major in initiation and progression of CRC; (5) Test can be potentially used on all pathogen groups; and (6) Microbiota dysbiosis induces methylation of host genes	(1) Complex bioinformatics analysis; (2) Expensive; (3) Microbiota composition depends on sample preparation, conservation, extraction protocol and many other factors; (4) Need a healthy control group; (5) Many microorganisms (virus, bacteria, fungi) have not been identified and sequenced; (6) Metabarcoding analysis provides only taxonomic affiliation based in small region; and (7) Analysis results depends on reference database

CRC: Colorectal cancer; ddPCR: Droplet digital polymerase chain reaction; FIT: Fecal immunochemical test; FOBT: Fecal occult blood test; FS: Flexible sigmoidoscopy; MT-sDNA: Multi-target stool DNA; TC: Total colonoscopy; TMB: Tumor mutational burden; WES: Whole exome sequencing; WGS: Whole genome sequencing.