Next-generation sequencing and its clinical application

NGS1-4 is a new technology for DNA and RNA sequencing and variant/mutation detection. This technology combines the advantages of unique sequencing chemistries, different sequencing matrices, and bioinformatics technology. Such a combination allows a massive parallel sequencing of various lengths of DNA or RNA sequences or even whole genome within a relatively short period of time. It is a revolutionary sequencing technology after Sanger sequencing5. NGS involves several major steps in sequencing. For example, DNA NGS involves DNA fragmentation, library preparation, massive parallel sequencing, bioinformatics analysis, and variant/mutation annotation and interpretation. DNA fragmentation DNA fragmentation is used to break the targeted DNA into many short segments, usually 100–300 bp in length. Different methods can be used to achieve this. DNA can be fragmented using mechanical methods, enzymatic digestion6, or other methods. For example, sonication can be used to break DNA into short segments. The short segments relevant to the targeted DNA sequences are pulled out using specific complementary probes of different designs7,8. This method is usually referred to as hybridization capture assay. Another method involves polymerase chain reaction (PCR) amplification. In this method, many pairs of primers are used to amplify the targeted DNA segments using PCR. The PCR products serve as short segments of targeted DNA. This method is usually called amplicon assay9,10. The DNA segments are then used for library preparation.