The rhAmpSeq Library Kit contains 2 amplification mixes optimized for rapid preparation of highly specific, sequencing-ready amplicon libraries.
- rhAmpSeq Library Mix 1—optimized for high specificity and multiplexing capability. This mix is for Targeted rhAmp PCR 1 in the rhAmpSeq workflow (Figure 1).
- rhAmpSeq Library Mix 2—optimized for high-efficiency amplification. This mix is for Indexing PCR 2 in the rhAmpSeq workflow. In this universal PCR step, rhAmpSeq Index Primers add index barcodes and
Illumina P5/P7 adapter sequences to the library (Figure 1).
Harnessing the power of rhAmp PCR
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.
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.
Table 1. rhAmpSeq system features and specifications.
|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.
Table 2. Choose the best rhAmpSeq library preparation protocol for your needs.
* Estimated time to process 12–96 samples using manual pipetting, including reaction setup, cleanup, library quantification, and normalization steps
|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|