Targeted enrichment of genomic DNA regions for next-generation sequencing

Abstract

In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.

Figure 1:

Commonly used targeted enrichment techniques. (1) Hybrid capture targeted enrichment either on solid support-like microarrays (a) or in solution (b). A shot-gun fragment library is prepared and hybridized against a library containing the target sequence. After hybridization (and bead coupling) nontarget sequences are washed away, the enriched sample can be eluted and further processed for sequencing. (2) Enrichment by MIPs which are composed of a universal sequence (blue) flanked by target-specific sequences. MIPs are hybridized to the region of interest, followed by a gap filling reaction and ligation to produce closed circles. The classical MIPs are hybridized to mechanically sheared DNA (a), the Selector Probe technique uses a restriction enzyme cocktail to fragment the DNA and the probes are adapted to the restriction pattern (b). (3) Targeted enrichment by differing PCR approaches. Typical PCR with single-tube per fragment assay (a), multiplex PCR assay with up to 50 fragments (b) and RainDance micro droplet PCR with up to 20 000 unique primer pairs (c) utilized for targeted enrichment.

Figure 2:

Comparison of enrichment factor calculations on sequencing depth and percent on target sequences for different target region sizes employed for targeted enrichment. Calculations were performed as follows: [52] EF, enrichment factor; ROI, region of interest in kb; genome, genome size in kb; pot, percent on target sequences; seq per run, assumed sequences per run in kb.

Figure 3:

Sequencing costs for short read next-generation sequencing. Since introduction in early 2008, costs dropped radically and are represented in a straight line on a logarithmic scale. The cost differential between sequencing, e.g. a full human genome or a human exome (plotted with already doubled coverage) is the cost that can be spent for targeted enrichment. Therefore, targeted enrichment is still an overall cost-efficient method, if costs for targeted enrichment stays within this cost space, this especially holds true for small target regions. Data adapted from NHGRI [51].

Figure 4:

Comparison of different enrichment techniques for regions of interest. The graph shows enrichments for the genes CTNNB1 (1), PT53 (2) and ADAM10 (3) converted to wiggle format on the UCSC genome browser. For every targeted enrichment experiment, the upper graph indicates the coverage obtained after data analysis, the corresponding bar line below specifies the region of interest used for probe design. Enrichment was performed with hybridization-based techniques either in solution (home made, blue; SureSelect whole exome, magenta; FlexGen, green) or on solid support (NimbleGen, yellow), with a PCR-based approach (RainDance, orange) and a circularization method (SelectorProbes, light blue).