Targeted sequencing is the application where researchers choose to only study a section of the genome and they do so by enriching specific genes, exons and/or other genomic regions of interest. This allows sequencing reads to be dedicated only to those specific areas, resulting in time and cost savings.1,2,3 Several steps are involved in the successful conversion of a precious sample into a sequencing-ready library. An additional step called target enrichment, which involves enriching specific target regions, can be added to this sample preparation workflow. Also known as targeted capture, this step provides a powerful way to study gene expression and to detect mutations, including structural variants and copy number variations.
The most frequently used enrichment methods for next-generation sequencing (NGS) are hybridization or capture-based target enrichment and amplicon-based target enrichment. While capture-based method uses specific probes for enrichment, amplicon-based method uses the power of PCR to do the same. There are advantages and disadvantages to both methods; choosing the right method may depend on the type of application where it is used. A new innovative method based on Primer Extension Target Enrichment (PETE) aims to combine the advantages of capture-based and amplicon-based target enrichment by offering high performance with speed and simplicity.
Hybridization of target regions can occur either on a solid surface (microarray) or in solution. The array-based method has several shortcomings, such as high cost, limitations to the number of samples and large sample requirements. The solution-based method overcomes these limitations, while retaining its advantages such as being scalable and easily automatable.
In the solution-based method, genomic DNA is first sheared into randomly sized fragments with mechanical or enzymatic DNA fragmentation. Sequencer-specific adapters carrying sample-specific barcode sequences are then added via a ligation reaction. A pool of biotinylated oligonucleotide probes targeting the desired genes, exons, and/or other genomic regions of interest is added to adapter-ligated DNA in solution for hybridization with targeted regions of interest. The hybridized probes are then captured and purified by streptavidin-coated magnetic beads and subsequently amplified and sequenced.