Custom rhAmpSeq Panels

Achieve deep, targeted amplicon sequencing with highly multiplexed panels

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.

Design custom panels for a wide range of plant and animal species, including human, mouse, tomato, corn, and grape
Multiplex up to 5000-plex in a single pool for efficient amplicon library prep
Choose from 3 delivery scales and formats for unparalleled flexibility to meet your project goals

Ordering

Custom rhAmpSeq Panels

Create custom NGS amplicon panels.

Design and order

To complete your rhAmpSeq workflow, you will also need the rhAmpSeq Library Kit and rhAmpSeq Index Primers. Additional details about the rhAmpSeq workflow can be found on the overview page.

Product details

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:

Marker-assisted selection and genomic selection in agricultural genetics and breeding
Confirmation of on-target and off-target CRISPR gene editing experiments
Hotspot panels for oncology, rare and inherited diseases, and other disease research

Design custom panels tailored to your research

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.

Figure 1. Overview of the rhAmpSeq Design Tool workflow.

Custom rhAmpSeq design

Custom assay design involves 2 key steps that provide improved performance over other amplicon sequencing systems:

Assay design involves extensive target site review and rhAmp primer design for each target, followed by comprehensive QC of each rhAmp primer to mitigate off-target effects, such as primer-dimer formation and non-specific genomic hybridization.
Virtual assay pooling is a unique multiplex primer QC feature that further enhances the specificity of rhAmp PCR technology. It allows us to more accurately predict potential primer-dimer amplification products that could lower overall reaction efficiency and decrease the percentage of correctly mapped reads.

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.

Technical details

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
			Panel size	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
			750	3000	30,000	60,000
			500	2000	20,000	40,000
101-499–plex	100 nM each primer	x nmol of primer/0.0002 nmol per rxn	400	2000	20,000	40,000
101-499–plex	100 nM each primer	x nmol of primer/0.0002 nmol per rxn	200	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
			50	800	8000	16,000
			25	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.

Performance

High quality sequencing data

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.