Custom rhAmpSeq Panels consist of rhAmp primer pairs designed to minimize primer dimers and maximize multiplexing capability for high quality amplicon libraries. This combination of robust, uniform coverage and efficient library prep allows you to confidently analyze more samples, faster. Use our rhAmpSeq Design Tool to easily input your targets and quickly receive your panels to start your targeted sequencing studies.
Custom rhAmpSeq Panels are highly multiplexed primer sets for targeted sequencing. Panels have been designed for and successfully tested in a wide range of animal and plant species, including human, mouse, tomato, corn, and grape.
Custom rhAmpSeq Panels are well suited for diverse targeted NGS applications, including:
Our rhAmpSeq Design Tool will allow you to quickly and easily design Custom rhAmpSeq Panels for your application and targets of interest (Figure 1). If you need additional help, our expert bioinformatics team can help custom-tailor a design for your specific project.
Once your design is complete, we provide a detailed design summary report for your custom panel so you can review the results and, if necessary, iterate your design before ordering your Custom rhAmpSeq Panel. You can also download assay BED files and a file containing assay IDs to order any sub-panel from the set of assays that were designed.
Custom assay design involves 2 key steps that provide improved performance over other amplicon sequencing systems:
Any assays that are successfully designed but not able to be pooled into the primary panel will be placed in secondary panels. These secondary panels have a designated prefix, such as P2 or SC, on the resulting panel name that the user entered during design submission. Refer to the rhAmpSeq Design Tool User Guide for more information.
Custom rhAmpSeq Panels are available in 3 convenient scales to fit your experimental needs: 0.4 nmol, 4 nmol, and 8 nmol. As shown in Table 1, we recommend adjusting rhAmp primer concentrations in the first amplification reaction (Targeted rhAmp PCR 1) depending on your panel size (plexity). Table 1 shows the approximate number of reactions for each scale based on panel size and scale.
Table 1. Number of rhAmp primer reactions based on scale and panel size.
|Panel size||rhAmp primer concentration in 10X panel*||Number of reactions per rhAmp primer order||Example|
|Panel size||Number of reactions|
|0.4 nmol primer (x)||4 nmol primer (x)||8 nmol primer (x)|
|≥500-plex||50 µM total primers||x nmol of primer/(0.1 nmol total/panel size)||1000||4000||40,000||80,000|
|101-499–plex||100 nM each primer||x nmol of primer/0.0002 nmol per rxn||400||2000||20,000||40,000|
|≤100-plex||250 nM each primer||x nmol of primer/0.0005 nmol per rxn||100||800||8000||16,000|
* Important: When creating rhAmpSeq primer pools, do not to combine forward and reverse primers for long term storage. The primer concentrations in the 10X panel stocks assume making a separate forward pool of primers and a separate reverse pool of primers. Refer to the protocols in the Resources section for details.
The data shown in Figure 2 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. Performance of your custom panel may depend on several factors, including the quality of the input sequences and the reference genome in the case of non-human species.
Figure 2. 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.
For more information, refer to the analysis guidelines in the User guides and protocols section.