What is the rhAmpSeq™ CRISPR Analysis System?
The rhAmpSeq™ CRISPR Analysis System is an advanced data analysis pipeline from Integrated DNA Technologies (IDT) for quantifying your genome editing results. Expect quick and accurate quantification of CRISPR-Cas edits. IDT's proprietary RNase H2-dependent PCR technology generates amplicon libraries for targeted sequencing on Illumina® NGS platforms. The system includes an advanced, yet accessible, cloud-based data analysis pipeline for quantification of on- and off-target edits.
Your advantages with this CRISPR Analysis System
- Best-in-class insertion/deletion (indel) quantification of genome editing
- Batch analysis of your results (multiplex up to 500 targets)
- High sequencing coverage uniformity and percentage of mapped reads
- Intuitive data analysis that requires no bioinformatics expertise
- Sample-to-analysis in under a week
Harnessing the power of rhAmp™ PCR for characterizing on- and off-target editing
The rhAmpSeq CRISPR Analysis System depends on IDT's proprietary rhAmp™ PCR technology. Using RNA-base-containing blocked primers (rhAmp primers), this technology harnesses the intrinsic specificity of the RNase H2 enzyme to cleave DNA:RNA duplexes. Due to its inherent mitigation of primer dimers, rhAmp PCR exhibits high specificity, even in multiplex reactions. The rhAmpSeq system combines this innate advantage with a convenient workflow that requires only two PCR 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 step (Targeted rhAmp PCR 1). Coupled with IDT's industry-leading oligo manufacturing process, the rhAmpSeq system offers fast and cost-effective analysis of your CRISPR results. Advantages of rhAmp PCR include an increased target affinity compared to traditional PCR, less off-target amplification, and a reduction of primer-dimers.
Workflow for analyzing your CRISPR-Cas editing results
Before you begin: Identify and nominate both on- and off-target sequences. The rhAmpSeq Analysis System workflow begins after the identification of potential targets. Off-target sites can be nominated using empirical methods such as GUIDE-Seq with or without in silico prediction tools.
Design and order your panel
Build your custom panels online. rhAmpSeq Panels have been specifically designed to assess CRISPR edits and are ideal for confirming on- and off-target CRISPR gene editing experiments. The panels are highly multiplexed primer sets for targeted sequencing. They have been expressly designed for a wide range of species. The rhAmp primer pairs in rhAmpSeq CRISPR Panels mitigate primer dimers and maximize multiplexing to provide you with high-quality amplicon libraries. Analyze more samples faster with the confidence of uniform coverage and the time savings of efficient library prep. IDT's rhAmpSeq™ Design Tool lets you quickly and easily design custom rhAmpSeq CRISPR Panels specific to your application and targets of interest. Once your design is complete, IDT provides a detailed summary report of the custom panel results for you to review and, when necessary, iterate your design before you order your custom rhAmpSeq CRISPR Panel. Also available for download are the associated assay BED files and assay IDs to order any sub-panel from the set of assays that were a part of your custom design.
Prepare your amplicon library
The rhAmpSeq CRISPR Library Kit is a rapid, cost-effective library preparation for rhAmpSeq CRISPR targeted sequencing. The kit contains two amplification mixes prepared for rapid, targeted, sequencing-ready amplicon libraries for Targeted rhAmp PCR 1 and Indexing PCR 2 in the rhAmpSeq workflow (Figure 1). Furthermore, the rhAmpSeq CRISPR Library Kit comes with analysis credits to enable data processing and quantification of editing events via the rhAmpSeq™ CRISPR Analysis Tool. Each analysis credit allows for the analysis of up to 500 targets for one, indexed sample.
rhAmpSeq Index Primers are used in the second amplification step of the rhAmpSeq™ workflow, Indexing PCR 2 (Figure 1), to add both unique index sequences and the P5/P7 sequences recognized by Illumina® sequencing instruments. These 96 index sequences are available for both the P5 and P7 primers. Adding these sequences to the amplicons created in the first amplification step creates dual-indexed libraries. This process enables combining rhAmpSeq amplicon libraries in a single sequencing run for maximum efficiency.
Sequence
Sequence your library on an Illumina® platform. Our partners will sequence the sample libraries on the Illumina® NextSeq platform to achieve your required coverage and read depth.
Analyze
IDT's rhAmpSeq CRISPR Analysis Tool is a flexible, cloud-based tool for interrogation of CRISPR-mediated, double-strand breaks. Benefit from the research-friendly user experience and obtain publication-ready data using our advanced data analysis pipeline without the need for coding or advanced bioinformatics experience. You gain access to the tool with purchase of rhAmpSeq CRISPR Library Kit.
Additional ressources
For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.