Targeted sequencing offers unique insights into specific regions of interest in the genome. It is a powerful application for investigating a variety of disease areas, such as oncology, inherited diseases, immunology and infectious diseases. This application allows targeting of specific genes, coding regions, even segments of chromosomes with precision and efficiency.
Targeted sequencing is more cost-effective than whole genome sequencing (WGS). It also enables deeper analysis of results than WGS and other survey approaches. In addition, it allows for deeper sequencing, and the depth of coverage helps in avoiding false interpretations of sequencing data. Because of this sensitivity, targeted sequencing provides tremendous advantage in variant calling in cancer research, identification of disease-associated mutations, single gene disorders and in gene expression studies. Targeted sequencing of specific regions also enables the discovery of causative genes for rare diseases. The focused approach of targeted sequencing provides the possibility of its use in targeted therapy applications and in personalized medicine efforts. For example, targeted resequencing of the polymorphic human leucocyte antigen (HLA) gene helps in HLA typing, which is crucial for matching in hematopoietic stem cell or solid organ transplantation.
The sample preparation workflow for targeted sequencing requires an additional step of target enrichment. It uses user-defined probe sets to enrich specific genomic regions of interest, thus causing only that region to be sequenced. The two methods for target enrichment are based on hybridization or amplification. While hybridization-based method uses probes to capture regions of interest, amplicon-based method uses PCR for target enrichment.
In the hybrid capture method, the process starts as a standard library preparation workflow. DNA is fragmented by shearing or using enzymes. Then adapters specific for the sequencing platform are added. Next, they are incubated with pools of biotinylated oligonucleotide probes designed to target specific regions of interest within a DNA fragment library. Finally, streptavidin-coated magnetic beads are used to attract the biotinylated probe/target hybrids. This method results in a sequencing-ready library that is highly enriched for the targeted DNA.
In PCR-based methods, both uniplex and multiplex PCR reactions can be used. In multiplex PCR several primers targeted toward different target genes are used to generate multiple amplicons in a single reaction. After amplification, a normalization step is carried out for normalizing the concentration of the multiple PCR products. Then the pooled products are sequenced. While this method is efficient and easy to use, it is not ideal for targeting large genomic regions due to the cost of reagents for multiple reactions. Failure of targets to amplify and PCR bias are other drawbacks associated with PCR-based enrichment methods.
Robust target enrichment and construction of libraries with maximum molecular complexity and minimal bias is critical for targeted sequencing applications. Roche offers performance-optimized hybridization-based probes, both as fixed designs and custom panels.
In addition, Roche also offers an integrated approach to sample preparation using its validated sample preparation solutions encompassing all the steps required (from sample collection to quantification) to convert a sample to a sequencing-ready library.