Advances in Targeted Region Enrichment Technologies

What is targeted region sequencing?

Whole genome sequencing looks at the whole genome of an organism, while targeted region sequencing looks at only the parts that are of interest. Targeted genome sequencing is a very sensitive and specific way to find SNPs, indels, CNVs, and large SVs. Compared to whole genome sequencing, targeted genome sequencing is more focused, economical, and effective. Furthermore, targeted regions can be sequenced at a much greater depth for a lower cost, which contributes to detecting rare variations. Whole exome sequencing is an example of targeted genome sequencing since it focuses on the exons. And using a targeted, or "hot-spot" panel, we can sequence an array of genes of interest. These genes may harbor mutations that are associated with the pathogenesis of diseases, or these genes may be clinically actionable genes of interest. This has been applied to biological and medical research, as well as clinical care. Here we mainly discuss the current target enrichment techniques and recommend qualified products.

Classical techniques for targeted enrichment

Target enrichment greatly facilitates the development of targeted region sequencing, making it affordable and efficient for complex genomes. There are three classical strategies for targeted enrichment.

Hybrid capture

The shotgun fragment library is set up so that it can combine with a library of DNA fragments that match the target regions. This can be performed on microarrays or in solutions. Mamanova et al. (2010) found that for small target sizes (approximately 3.5 Mb), solution capture yields superior coverage of the target regions than an array-based approach, and that for whole exome enrichment, both approaches perform equally. We recommend SureSelect (Agilent Technologies), Nextera (Illumina), TruSeq (Illumina), and SeqCap EZ (Roche NimbleGen) for in-solution hybridization-based target enrichment.

Selective circularization

Molecular inversion probes (MIPs) consist of a common sequence flanked by target-specific sequences. After the hybridization in the regions of interest, MIP is subjected to a gap-filling reaction and ligation to generate closed circles. The MIPs can be hybridized to mechanically sheared DNA, while selector probes use a restriction enzyme cocktail to digest the DNA, and the probes are adapted to the restriction pattern. MIPs and selectors share a common central linker, which can include the sequencing primer for NGS. Therefore, NGS library construction is not required. We recommend HaloPlex (Agilent Technologies) for selective circularization-based target enrichment.

PCR amplification

Multiple long-range PCRs, multiplex PCRs, and microdroplet PCRs are all ways that PCR is used to enrich regions of interest. There are many products for long-range PCR, such as SequalPrep (Thermo Fisher Scientific) and SeqTarget (Qiagen), Ion Ampliseq (based on high-multiplex PCR), and RainDance's microdroplet technology.

The comparisons of classical target enrichment techniques

Each of the four strategies has its own merits and demerits. For example, the specificity of PCR exceeds that of hybrid capture, but its uniformity is not matched by either MIPs or hybrid capture.

Advanced method: Region-Specific Extraction (RSE)

The traditional methods heavily rely on fragmentation of DNA prior to amplification, which generates relatively short sequences (less than 1kb). Because they could not provide fragments large enough to span confounding sequence elements, this is a limiting factor for characterizing complex genomic loci. Fortunately, the enrichment method — Region Specific Extraction (RSE), proposed by Dapprich et al. (2016), addresses this unmet need by capturing long DNA fragments (approximately 20kb).

The principle of RSE is outlined in Figure 2. Briefly, the genomic DNA is denatured to be hybridized with capture primers. The bound primers are then enzymatically extended with incorporated biotinylated dNTPs. The targeted DNA segments are pulled down by streptavidin-coated magnetic particles. The captured DNA can be amplified by whole-genome amplification and subsequently processed by next-generation sequencing.

Metrics for the assessment of a target-enrichment experiment

There are several metrics that need to be considered in target-enrichment experiments. To deal with this issue, a universal set of Target Enrichment Sequencing Descriptors (TESD) metrics has been developed, enabling the assessment of target enrichment results. The TESD uses the following parameters:

Region of interest, ROI

Average read depth in region of read, DROI

Specificity, S

Enrichment factor, EF

Evenness, E

Weight of input DNA requirement, W

Fraction sufficiently covered at a read depth of x, Fx

Of the seven metrics, ROI and W determine the input requirement, and the other five provide a measurable report on either the enrichment (S, EF, E) or the sequencing results (DROI, Fx).