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Amplicon sequencing

Overview

Amplicon sequencing is a type of targeted next generation sequencing that uses PCR to create DNA sequences called amplicons. It is simple and fast with a variety of applications.

What is amplicon sequencing?

Amplicon sequencing is a method of targeted next generation sequencing (NGS) that enables you to analyze genetic variation in specific genomic regions. This method uses PCR to create sequences of DNA called amplicons. Multiplexing – barcoding samples so that they can be mixed into pools – allows multiple samples to be sequenced on a single sequencing run. Before multiplexing, individual samples used for amplicon sequencing must be transformed into libraries by adding adapters and enriching target regions by PCR amplification. The adapters allow formation of indexed amplicons and enable the amplicons to adhere to the sequencing flow cell.

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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.

Simple and fast with high on-target rates

Amplicon sequencing is a quick and easy targeted sequencing method with a simple workflow and fast turnaround time, as compared to hybrid-capture sequencing. Amplicon sequencing has naturally higher on-target rates than other targeted sequencing methods due to the precision of primer design.

This method is appropriate for genotyping by sequencing, identification of germline single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and known fusions. Amplicon sequencing should be used for experiments that can tolerate more false positive variant calls, since bias that might be created during the PCR amplification step cannot be removed.

Advantages of amplicon sequencing

Listed are many reasons why amplicon sequencing might be the right choice for your research:

  • Reduces sequencing costs and turnaround time compared to other approaches, like WGS
  • Low DNA input requirements while providing higher on-target rate compared to hybridization capture
  • Facilitates efficient discovery and screening of genomic variants with a highly targeted approach
  • Supports multiplexing of 100’s–1000’s of amplicons in a single reaction for high coverage
  • Delivers highly targeted resequencing
  • Amenable to a wide range of experimental designs, such as allelic variant identification, structural variant and breakpoint identification, marker gene sequencing for microbiome studies, and more

Whether you need custom or predesigned hybridization panels, exomes or targeted amplicons, IDT has the solution you need. Explore our product offerings in hybridization capture and NGS amplicon sequencing.

Amplicon sequencing for rapid SARS-CoV-2 variant identification

Rapid, accurate pathogen identification is critical for controlling outbreaks and pandemics, such as the current COVID-19 pandemic. With xGen™ SARS-CoV-2 Amplicon Panels (previously Swift Normalase™ Amplicon Panel (SNAP) SARS-CoV-2), researchers can go from cDNA to sequencer in only three hours.

SARS-CoV-2 variants can be identified from an array of sample types, including nasopharyngeal/oropharyngeal swabs, sputa, stool, and wastewater. The panel covers 99.7% of the SARS-CoV-2 genome for identification of mutations from alpha, delta, and omicron strains.

Table 1. NGS for SARS-CoV-2 specifications.

FeaturesSpecifications
Design coverage and panel information
  • 99.7% (29,828 of 29,903 total bases)
  • 345 amplicons, sized 116-255 bp (average 150 bp)
Input material
  • 1st or 2nd strand cDNA
  • Minimum 10 – 100+ viral copies (qRT- PCR Ct value 30-40)
Time2 hours cDNA-to-Library
3 hours cDNA-to-Normalized-Library Pool
Multiplexing capability
  • Up to 384 CDIs
  • Up to 1,536 UDIs
Compatible with other indexes?Yes
Recommended depth
  • Identification: 50K reads/library
  • Variant calling: 1M reads per library

For more information on xGen SARS-CoV-2 Panels and how to use them in your research, visit our Product Page.

Get started with amplicon sequencing

Whether your workflow requires rapid turnaround, or starts with low biomass samples, we can provide the solution. Learn more about xGen NGS amplicon sequencing and how it can meet your research needs.

Amplicon sequencing resources

Explore our expert-curated amplicon sequencing resources from primary literature to IDT’s own collection of DECODED articles.

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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