Use the rhAmpSeq Library Kit with rhAmpSeq panels and rhAmpSeq Index Primers to quickly prepare amplicon libraries for sequencing on Illumina platforms. Our proprietary rhAmp PCR technology enables a simple, elegant workflow with only 2 PCR master mixes for optimum specificity and high multiplex amplification capability.
The rhAmpSeq Library Kit contains 2 amplification mixes optimized for rapid preparation of highly specific, sequencing-ready amplicon libraries.
Our proprietary rhAmp PCR technology drives the rhAmpSeq system. This technology harnesses the intrinsic properties of the RNase H2 enzyme and RNA-base–containing blocked primers (rhAmp primers), minimizing primer dimers and enabling rhAmpSeq panels to be highly multiplexed.
Those 2 advantages—minimal primer dimers and high multiplexing capabilities—allowed us to develop the high-performance rhAmpSeq system, whose fast, easy workflow requires only 2 PCR amplification steps (Figure 1) to generate amplicon libraries for Illumina platform sequencing.
To learn more about rhAmp PCR technology, visit this page.
Figure 1. Detail of 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 barcodes and P5/P7 sequences are incorporated during Indexing PCR 2.
Because all rhAmpSeq reagents are compatible with both our regular and high-throughput library preparation protocols, you can choose the best workflow for each experiment without having to buy different reagents. Table 1 shows the features and specifications common to both rhAmpSeq system protocols.
|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)|
When multiplexing many samples in a single NGS run, we have observed slightly better sample-level coverage uniformity with the regular rhAmpSeq library preparation protocol. Nevertheless, the high-throughput protocol also offers effective genotype calling, and performs best when read coverage is not limiting (e.g., >500X per target).
In contrast to the regular protocol, the high-throughput protocol saves both overall time and reagent costs by removing a cleanup step and the need to quantify and normalize libraries before combining libraries onto a flow cell. However, your results may vary—please contact Application Support for more information.
|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|
Your sequencing data can only be as good as your amplicon libraries. High quality amplicon library preparation reagents are therefore essential for obtaining excellent and reproducible performance. The data in Figure 2 show typical consistency and performance of the rhAmpSeq Library Kit.
Figure 2. Multiple rhAmpSeq Library Mix lots demonstrate consistent performance. We evaluated the performance of 3 lots of rhAmpSeq Library Mix. Each lot was evaluated with multiple Coriell samples (10 and 50 ng input DNA) using the rhAmpSeq Sample ID Panel following the high-throughput rhAmpSeq library preparation protocol (32 replicates per input quantity per lot). Performance was consistent across lots. Coverage uniformity is the percent of targets with coverage ≥0.2X of the mean.
For more information, refer to the analysis guidelines in the User guides and protocols section.