Targeted sequencing focuses next generation sequencing
Next generation sequencing has revolutionized genomics. High-throughput sequencing enables sequence profiling of DNA and RNA, and can run samples in parallel, resulting in massive time savings. The ability to generate large amounts of sequence data in a relatively short amount of time supports a wide range of genetic analysis applications and accelerates advances in research, clinical, and applied markets.
To even further decrease time and cost associated with sequencing, targeted NGS allows users to sequence specific areas of the genome for in-depth analyses, instead of sequencing the whole genome (WGS). Targeted sequencing can identify known and novel variants within your region of interest for both discovery and diagnostics. The method generally requires less sample input, produces a smaller amount of data than WGS, and makes analyses more manageable.
Types of targeted sequencing
There are several methods of targeted sequencing, each appropriate for specific applications. The most popular methods are hybridization capture, amplicon sequencing, and molecular inversion probes (MIPs). The technology behind these methods are all different, and come with their own benefits and challenges. IDT provides solutions for hybridization capture and amplicon sequencing, which are compared below (Table 1).
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|
|Sensitivity||Down to 1% without UMIs||Down to 5%|
|Total time||More time||Less time|
|Cost per sample||Varies||Generally lower cost per sample|
Genotyping by sequencing
Considerations when choosing a targeted sequencing method
The method chosen for performing targeted sequencing can be determined by considering several factors:
Total time and level of automation: Amplicon sequencing can be completed in the shortest amount of time.
Throughput: Hybridization capture allows more targets to be sequenced per panel.
Target rate and uniformity: Amplicon sequencing has naturally higher on-target rates than hybridization capture due to the specificity of primer design. This comes at the cost of uniformity.
Targeted sequencing applications
Targeted sequencing can be used for a variety of applications. Hybridization capture works well for genotyping and rare variant detection. It is the method of choice for exome sequencing and is commonly used in oncology research, both for discovery and diagnostics. Amplicon sequencing is used for genotyping by sequencing and for detection of germline SNPs, indels, and known fusions. It is particularly suited to detection of disease-associated variants, as well as validating CRISPR on- and off-target edits after genome editing (See Evaluate CRISPR-Cas9 edits quickly and accurately with rhAmpSeq targeted sequencing).
All of these points are examined in detail with recommendations and rationale provided in the Targeted sequencing guide. This extensive application guide provides chapters on assay selection and application, time requirements, and throughput. Target rate and uniformity are also discussed. The guide is written by IDT scientists, and is free—simply download it here.