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Targeted next generation sequencing

Overview

Targeted next generation sequencing is a type of next generation sequencing (NGS) that focuses on specific areas of the genome. Popular methods of targeted NGS include hybridization capture and amplicon sequencing.

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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.

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Targeted sequencing application guide

This detailed overview walks you through major advances in capture and enrichment technology, types of targeted next generation sequencing, their applications, and more.

Table 1. Comparison of targeted sequencing methods.

FeatureHybridization captureAmplicon sequencing
Input amount1–250 ng for library prep, 500 ng of library into capture10–100 ng
Number of stepsMore stepsFewer steps
Number of targets per panelVirtually unlimited by panel sizeFewer than 10,000 amplicons
SensitivityDown to 1% without UMIsDown to 5%
Total timeMore timeLess time
Cost per sampleVaries Generally lower cost per sample
Best-suited applications

Exome sequencing
Genotyping
Oncology
Detecting rare variants
Gene discovery
Detecting low-frequency somatic SNVs* and indels

Genotyping by sequencing
Detecting CRISPR editing events
Detecting disease-associated variants
Detecting germline inherited SNPs** and indels

*single nucleotide variant **single nucleotide polymorphism
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xGen Exome Research Panel v2 white paper

Learn how our large-scale production platform, using PCR-free synthesis, provides a unique advantage over array-based platforms by delivering consistent exome panel performance over time.

Products for targeted sequencing solutions

Working in one of these applications? Just starting? See how you can easily improve your workflows and results.

NGS Discovery Pools

NGS Discovery Pools allow you to build custom panels quickly at a fraction of the cost of conventional custom panels.

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xGen Lockdown Panels

xGen Lockdown Panels are stocked enrichment panels for targeted next generation sequencing. The panels consist of individually synthesized and quality-controlled xGen Lockdown Probes that have been validated to provide the highest level of performance.

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xGen Exome Research Panel v2

The xGen Exome Research Panel v2 consists of 415,115 individually synthesized and quality controlled xGen Lockdown Probes. The Exome Research Panel spans a 34 Mb target region (19,433 genes) of the human genome and covers 39 Mb of end-to-end tiled probe space.

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rhAmpSeq amplicon sequencing system

The rhAmpSeq system enables highly accurate amplicon sequencing on Illumina next generation sequencing (NGS) platforms. Whether you are investigating thousands of targets or a few, the fast and easy rhAmpSeq workflow generates NGS-ready amplicon libraries for deep, targeted resequencing.

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