What is targeted next generation sequencing (NGS)?
Targeted next generation sequencing allows you to sequence specific areas of the genome for in-depth analyses more rapidly and cost-effectively than whole genome sequencing (WGS). Targeted sequencing uses deep sequencing to detect known and novel variants within your region of interest. This method generally requires less sample input and produces a smaller amount of data than WGS, making analyses more manageable.
What are the methods of targeted NGS?
There are several methods of targeted sequencing, each appropriate for specific applications. The most popular methods are hybridization capture and amplicon sequencing (Table 1).
The main difference between techniques is the method by which the samples are enriched. Hybridization capture is performed in solution using biotinylated oligos (i.e., baits or probes) to capture complementary sequences from the sample library. Amplicon
sequencing uses PCR primers to amplify the sequences of interest.
Another differentiating factor is the point at which samples can be multiplexed. Multiplexing, also called pooling, allows multiple samples to be processed simultaneously, saving cost and time. 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. Additional indexes, called unique molecular identifiers (UMIs) can be used to identify specific molecules within a sample. Using adapters with UMIs in hybridization
capture allow you to remove PCR duplicates for better quantitation or use multiple duplicate reads for in silico error correction to increase accuracy by reducing the rate of false positives.
Note: PCR is used in some hybridization capture protocols during library preparation to increase DNA input of small samples.