Table2.

Advantages and disadvantages of DNA sequencing

Method	Basic technique	Advantages	Disadvantages	Reference
Sanger-chain termination method (first generation sequencing)	Fluorescent dye-labeled bases; DNA fragments separated by capillary electrophoresis	High sensitivity, gold standard complete sequence	Very time consuming; cannot detect deletions, translocations or copy number changes	133-136
Pyrosequencing-sequencing by synthesis method	Chemiluminescent detection; DNA polymerase synthesizes cDNA to a target template; pyrophosphate release is detected at each base addition	More sensitive than Sanger; provides % of mutated vs. wild-type DNA; works well with fragmented DNA	Short read length limits technique to hot spots. Limited accuracy at detecting changes in homopolymer runs. Scalability is limited compared with other NGS methods	136-140
Allele-specific RT-PCR	Primers span DNA sites of interest and probes detect specific mutations	Very high sensitivity widely used for clinical testing for oncogene mutations in CRC and NSCLC	Scalability constraints limit application to hot spots	136,141-143
RT-PCR melting curve analysis	Heterogeneous DNA PCR products melt at different temperatures than homogenous DNA/PCR products	High sensitivity provides percentage of mutated versus wild-type DNA	Often difficult to resolve differences in melt curves. Difficult to standardize. Multiplex capability is limited	136,144