Next-generation sequencing technology is used in targeted gene panels to explore the mutation status of multiple genomic regions of interest at the same time. Areas of the genome related to disease or phenotype of interest are included in the targeted panels. Traditional sequencing methods may skip point mutations, inserts and deletions, copy number variates, and translocations, but gene panels can help researchers find them.
Pre-designed or custom-designed targeted gene panels are available. Genes of clinical importance linked to cancer, Mendelian diseases, cardiac disorders, and neurodegenerative conditions are found in the most popular pre-gene panels. This method can be used to extract DNA from a variety of sample materials, such as formalin-fixed paraffin-embedded. Targeted gene panels help lessen sequencing time and streamline data analysis by concentrating only on key areas of interest.
Amplicon or capture-based targeted sequencing are the two main types of targeted sequencing. Prior to library preparation, amplicon-based enrichment uses specially designed primers to enhance only the areas of interest. In contrast, in capture-based methods, the DNA is fragmented, and targeted areas are enhanced using hybridization oligonucleotide bait sequences affixed to biotinylated probes, enabling isolation from the remaining genetic material. Amplicon-based enrichment is the less expensive of the two technologies and produces a higher number of on-target reads; however, hybrid sequencing provides more uniform coverage of these regions. Specific primers that can augment overlapping fragments in a single PCR reaction are used in some commercially available amplicon platforms to resolve coverage issues. Amplicon-based sequencing uses much less starting material than hybrid-capture sequencing, making it optimal for situations where there isn't enough DNA for TS.
Compared to amplicon enrichment, hybrid-capture has been demonstrated to generate fewer PCR duplicates. In comparison to the identical amplicons generated by amplicon enrichment platforms, the random shearing of DNA in hybrid-capture platforms lessens the risk of two unique fragments aligning to the same genomic coordinates, making these duplicates easier to remove computationally. This makes hybrid-capture particularly useful for specimens like FFPE and ctDNA where PCR artifacts are more likely to occur. Furthermore, certain areas of the genome make amplicon enrichment primer design difficult. The long bait sequences used in hybrid-capture, on the other hand, allow for more specific region selection. Overall, hybrid-capture-based platforms provide further accurate and uniform target selection, whereas amplicon-based platforms are frequently used in small-scale experiments where sample quantity or cost are important considerations.
There are a variety of methods for establishing gene panels. Target enrichment is frequently achieved through solution hybridization, in which probes pull down the genomic regions to which they are specific. Optionally, enrichment via PCR can be used, in which each targeted region is amplified in a multiplexed reaction using a different pair of primers.
Hybridization microarrays and alternative types of PCR multiplexing are two other methods for target enrichment. Thousands of DNA targets can be sequenced continuously using next-generation sequencing technologies, depending on the method used and the project requirements.
- Low-cost research for Mendelian disease and cancer clinical trials
- Classification of rare variants at low allele frequencies
- Detection of rare variants with low allele frequencies