Targeted next generation sequencing (NGS) focuses on specific regions of interest in the genome. With targeted NGS, researchers can target specific genes, coding regions, or even chromosomal segments at deeper coverage than alternative sequencing methods, obtaining fast, accurate, and precise genomic insights.
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:
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.
|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|
Genotyping by sequencing
*single nucleotide variant
**single nucleotide polymorphism
This detailed overview walks you through major advances in capture and enrichment technology, types of targeted next generation sequencing, their applications, and more.
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