Targeted sequencing handbook

The takeaway: Are you getting the most from your targeted sequencing efforts? Our new Targeted sequencing guide runs down what you need to know about hybridization capture and amplicon sequencing as you seek to streamline your NGS research.

Targeted sequencing, also sometimes called target amplicon sequencing, is a sequencing technique used in next generation sequencing that focuses on amplicons and specific genes. Gene targeted sequencing has a wide array of uses but is particularly helpful in population genetics as it can target a broad range of organisms. It is also useful in DNA studies since it permits the application of a specific gene area by polymerase chain reaction, or PCR.

Besides its applications, targeted RNA sequencing is gaining popularity because it uses shorter reads—usually 50 to 400 base pairs—which permit faster screening of multiple specimens and less expensive overall operations when compared to whole genome sequencing. Plus, since the method tends to produce less data than whole genome sequencing, both the sequencing and analysis operations are easier to conduct.

Targeted NGS has two main methods—hybridization capture and amplicon sequencing. Each method has its benefits, and these should be carefully considered by researchers. Looking for more help? IDT’s Targeted sequencing guide provides an introduction to hybridization capture and amplicon sequencing and can guide researchers as they seek more information on how to put these tools to use.

Using the Targeted sequencing guide, let’s look at these two NGS methods in more detail. 

Hybridization capture: Ideal for multigene panels

Hybridization capture is a targeted NGS method that uses long, biotinylated oligo probes to hybridize target regions. Its top uses are in genotyping, exome sequencing, and investigating rare variants.

Researchers using this method will first convert DNA samples into sequencing libraries, with regions of interest in the library captures using the long probes. After bait hybridization, a magnet is used to separate the streptavidin-biotin bind. The baits can be arranged in different ways to sequence the region of interest.

Features of hybridization capture include:

• Low DNA input
• Comparatively more complex and time-consuming workflow
• Comparatively more expensive
• Larger gene content
• Practically unlimited targets per panel
• Sensitivity to 1%

Applications of hybridization capture include: 

• Exome sequencing
• Genotyping
• Mutation discovery
• Oncology research
• Identification of rare variants

What is amplicon sequencing?

Amplicon sequencing is a fast tool for specific variant detection. Amplicon sequencing is a related NGS method that falls under the umbrella of targeted NGS use, though it has different features and uses. It is mostly used for oncology research, rare variant detection, and rare variant detection. 

In amplicon sequencing, a multiplexed PCR approach is used to create amplicon DNA molecules that are generated from target-specific primer pairs. The amplicons are then converted into libraries with NGFS adapters. Panel designs can vary significantly and primer design, while potentially difficult, can identify mutations and help provide critical insights.

Features of amplicon sequencing include:

• Increased DNA input
• Faster, simpler workflow
• Comparatively higher cost
• Smaller gene content 
• Fewer than 10,000 amplicons per panel 
• Sensitivity to 5%

Applications of amplicon sequencing include: 

• Oncology research
• Genotyping
• CRISPR confirmation
• Identification of specific variants
• Virology
• Metagenomics
• Identification of rare variants

Targeted sequencing vs. amplicon sequencing: Which is best for you?

While both of these sequencing methods have their benefits and drawbacks, which one you choose will likely hinge on what line of research is being pursued. As noted on the handbook, both methods can handle challenging samples, though damage to a sample may be a factor to consider.  Read depth requirements are also important to assess. Both methods can support a wide target size range, but amplicon panels are best for smaller target ranges and hybridization capture panels shine with larger ranges. Allele frequency is a key consideration as well; hyb capture panels can identify larger indels in DNAS and both known and unknown RNA fusions.

Key considerations include:

• Time: Amplicon sequencing can be completed in the shortest amount of time 
• Throughput: More targets can be enriched and sequenced per panel with hyb capture
• Target rate and uniformity: Hyb capture delivers better uniformity and complexity, though amplicon sequencing variants have higher on-target rates

Looking to learn more? As a trusted partner in the genomics industry, IDT’s Targeted sequencing guide is dedicated to helping researchers of all ability levels find the best technology and methods for their work. Simply click here to download your free copy of the guide.