rhAmpSeq Amplicon Sequencing

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rhAmpSeq amplicon sequencing system—NGS power. PCR simplicity.

Faster discovery with a rapid and accurate targeted sequencing solution

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, based on our proprietary RNase H2-dependent PCR (rhAmp PCR) technology, generates NGS-ready amplicon libraries for deep, targeted resequencing.
  • •Achieve cost-effective library preparation using custom or predesigned panels
  • •Reduce the formation of primer dimers or misprimed PCR products
  • •Perform only 2 PCR amplification steps in a fast, easy workflow
  • •Ensure robust results with IDT manufacturing quality


Harnessing the power of rhAmp PCR

Our proprietary rhAmp PCR technology drives the rhAmpSeq system. Using RNA-base–containing blocked primers (rhAmp primers), this technology harnesses the intrinsic properties of the RNase H2 enzyme to recognize and cleave DNA:RNA duplexes. rhAmp PCR is thus a powerful tool for increasing amplification specificity and minimizing the biggest limitation of PCR multiplexing—primer dimers.

The rhAmpSeq system combines the performance advantages of rhAmp PCR with a fast, easy-to-use workflow that requires only 2 PCR amplification steps (Figure 1) to generate amplicon libraries for Illumina sequencing platforms. Figure 1 shows both amplification steps in the rhAmpSeq workflow, highlighting the role of rhAmp PCR technology during the first amplification step (Targeted rhAmp PCR 1). Coupled with our best-in-class DNA and RNA manufacturing, the rhAmpSeq system offers high quality, targeted, sequencing results quickly and cost-effectively.

To learn more about rhAmp PCR technology, visit this page.


How rhAmpSeq PCRs work

Figure 1. Amplification steps in the rhAmpSeq workflow. RNase H2 activates rhAmp primers by target-specific cleavage of the RNA base within the DNA:RNA duplex, removing a 3′ blocker. RNase H2 activity is highly specific, thus reducing the amount of amplification from non-specific hybridization and primer dimers. Only activated rhAmp primers can be extended to generate target amplicons.

Illumina sample indexes and P5/P7 sequences are incorporated during the second amplification step.


 

A simple workflow with few components

The rhAmpSeq system offers a simple, easy workflow with only 2 PCR amplification steps, using either a custom or predesigned panel. To begin creating your amplicon libraries, simply choose a panel, then add the rhAmpSeq Library Kit and rhAmpSeq Index Primers.

Simple workflow with few components


 

Figure 2. Overview of the rhAmpSeq system workflow.



 

Amplicon library preparation that matches your throughput needs

The rhAmpSeq system can be used with either a regular or a high-throughput protocol. Both protocols are automation friendly. The regular protocol offers slightly higher NGS performance, while the high-throughput protocol reduces both overall workflow time and cost by removing cleanup steps and the need to quantify and normalize DNA libraries before pooling (Table 1). rhAmpSeq libraries are compatible with Illumina NGS platforms.

Table 1. Choose the best rhAmpSeq library preparation protocol for your needs.
 
Considerations Regular protocol High-throughput protocol
Better sample-to-sample coverage uniformity
Better performance with challenging sample types (e.g., FFPE, cfDNA)
Ideal for high-throughput screening labs
No library quantification and normalization required
Hands-on time* 2.5–4.5 hr 1–1.5 hr
Total workflow time* 4–6 hr 4–4.5 hr
* Estimated time to process 12–96 samples using manual pipetting, including reaction setup, cleanup, library quantification, and normalization steps

High-quality performance

The rhAmpSeq system offers consistent performance across panel sizes ranging from tens to thousands of amplicons in a single multiplex reaction (Figure 3), and supports a variety of applications, including challenging sample types [e.g., formalin-fixed paraffin-embedded (FFPE) and cell-free DNA (cfDNA) samples]. Table 2 summarizes key features, specifications, and performance metrics for the rhAmpSeq system.


The data shown in Figure 3 are representative of performance from 3 different custom rhAmpSeq panels using random SNP markers in the human genome with initial (non-optimized) rhAmpSeq panel designs. The performance of your custom panel may depend on several factors, including the quality of the input samples and the reference genome in the case of non-human species.
 

Table 2. rhAmpSeq system features and specifications.
Feature Specification
Supported protocols Regular library preparation (10–100 ng)
High-throughput library preparation (10–50 ng)
Sample type Tissue, FFPE, cfDNA
Insert size Flexible (50–200 nt)
Custom panel size Up to 5000 amplicons per panel
Sample indexing capability 96 index sequences (up to 9216 combinations)
Compatible platforms Illumina

High quality sequencing data
Figure 3. High quality sequencing data across a range of non-optimized panel sizes and DNA input quantities. Coriell DNA samples were used to evaluate the performance of non-optimized rhAmpSeq panels of varying sizes (20, 282, and 994 amplicons) following the high-throughput (HT) protocol using 10 and 50 ng of DNA input. The largest panel (994 amplicons) was evaluated following the regular (Reg) protocol with 10 and 100 ng of DNA input. Coverage uniformity is the percent of targets with coverage ≥0.2X of the mean. Error bars = standard deviation from the mean.



 

Faster discovery, fewer headaches

The rhAmpSeq system can be used in a wide variety of research applications, and should be considered for any application that requires stringent and efficient sequencing analysis. These are just a few of the fields in which rhAmpSeq technology has been successfully used.

Crops: rhAmpSeq AgBio applications

Agricultural biotechnology

Genotyping by sequencing is an agricultural biotechnology technique for molecular breeding and trait/marker selection. Whether you are focused on a few markers for marker-assisted selection or on thousands of markers for genomic selection, the rhAmpSeq system can help accelerate your plant or animal improvement programs.





 

CRISPR editing events: rhAmpSeq applications

CRISPR genome editing

CRISPR genome editing has recently become a dominant technology. However, CRISPR editing can produce unwanted off-target editing events. Custom rhAmpSeq Panels save time and resources by allowing you to quickly interrogate many CRISPR-edited sites simultaneously.
No more single PCRs!






 

Cells: rhAmpSeq disease research

Disease research and sample identification

Detecting disease-associated variants is a key element of human-focused research. With the rhAmpSeq system’s combined specificity and multiplexing capabilities, you can easily and accurately detect “hotspot” variants. In addition, the rhAmpSeq Sample ID Panel can be used to track samples and avoid mix-ups in human sample workflows.





 

Design custom panels tailored to your research

Need a custom panel? Our expert bioinformatics team will work with you to design custom-tailored rhAmpSeq panels for your species and targets of interest. After completing the design of your custom panel, we will send you a project summary to review, and a link to view and order your panel through our website.

Visit our rhAmpSeq custom panels page to begin your custom design request.