What is amplicon sequencing?
Amplicon sequencing is an experimental method of targeted next generation sequencing (NGS) that enables you to research the sequence of specific genomic regions. This method uses PCR to create copies of your targeted DNA sequences to create amplicons.
Unlike a typical PCR experiment with a single set of forward and reverse primers, amplicon sequencing panels include multiple sets of primer pairs to copy multiple regions of the DNA simultaneously. Individual research samples used for amplicon sequencing
must be transformed into libraries by adding adapters and enriching target regions to each of the amplicons with a second PCR amplification or adapter ligation. The adapters allow the amplicons to adhere to the flow cell or bead for NGS sequencing.
The amplicons from different research samples can be pooled (referred to as multiplexing) which involves adding a barcode or index sequence to the samples so they can be identified during data analysis. The adapters can either include the index sequences
or can be added during the PCR amplification step. Depending upon the number of amplicons in the targeted sequencing library, one lane of the sequencing flow cell can decode hundreds to thousands of indexed libraries.
As seen in Figure 1, xGenTM NGS Amplicon Sequencing technology uses multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries for research studies. The PCR1+PCR2 workflow generates
robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods such as Qubit® (Thermo Fisher) or Agilent Bioanalyzer® or normalized using the included
xGen Normalase™ reagents.
Key benefits include:
- Amplify hundreds to thousands of targets in a single tube PCR
- Amenable for low input samples such as cell-free DNA (cfDNA)
- Supports high throughput methods
- Can be designed for a variety of research applications including variant detection or targeted pathogen sequencing
- Compatible with Illumina® sequencing platforms with the correct adapter and index sequences