What is targeted sequencing?
Targeted next generation sequencing focuses on specific genomic areas of interest. This technology is ideal for examining genes in specific pathways or for follow-up experiments (targeted resequencing) from whole genome sequencing (WGS). It is rapid and more cost-effective than WGS, and because it allows for deeper sequencing. Targeted sequencing is an especially sensitive
and powerful method for identifying variants and mutations, including rare variants. Additional advantages of targeted NGS compared to WGS include:
- Smaller datasets requiring less computational resources
- More scalable (can handle more samples/sequencing run)
- More appropriate for industrial applications where cost and speed are critical
How does targeted sequencing work?
Targeted sequencing is similar to WGS, but the sample preparation workflow requires an extra step: target enrichment. The two main target enrichment methods are hybridization capture and amplicon sequencing.
Hybridization capture uses biotinylated oligonucleotide probes to capture regions of interest, while amplification uses PCR for target enrichment (see Table 1).
One major difference between the two approaches is the point at which samples can be multiplexed, or pooled. Multiplexing requires adding a barcode (index) to samples so they can be identified after sequencing. Samples used for hybridization capture can
be multiplexed after library preparation, but before target capture (enrichment). Samples used for amplicon sequencing must be transformed into libraries and enriched via PCR amplification individually before they can be multiplexed for sequencing.
When using hybridization capture, additional indexes, called unique molecular identifiers (UMIs) can be used to identify specific molecules within a sample. Using adapters with UMIs facilitates the removal of PCR duplicates for better quantitation or
the use of multiple duplicate reads for in silico error correction to reduce false positives and increase accuracy.
The appropriate method for target capture depends on several variables, including the desired accuracy, budgetary constraints, and the downstream sequencing application. Table 1 below can help you determine which targeted sequencing method is best for
your research applications.
Table 1. Comparison of targeted sequencing methods.
|Feature||Hybridization capture||Amplicon sequencing|
|Input amount||1–250 ng for library prep, 500 ng of library into capture||10–100 ng|
|Number of steps||More steps||Fewer steps|
|Number of targets per panel||Virtually unlimited by panel size||Fewer than 10,000 amplicons|
|Variant allele frequency sensitivity||Down to 1% without UMIs||Down to 5%|
|Total time||More time||Less time|
|Cost per sample||Varies ||Generally lower cost per sample|
Detecting rare variants
Detecting low-frequency somatic SNVs* and indels
Genotyping by sequencing
Detecting CRISPR editing events
Detecting disease-associated variants
Detecting germline inherited SNPs** and indels