rhAmpSeq Index Primers

Combine amplicon libraries in a single sequencing run for maximum efficiency

Use rhAmpSeq Index Primers to prepare amplicon libraries for targeted sequencing on Illumina platforms by adding a unique, identifying “barcode” sequence to each amplicon. Request exactly the primers you need from the 96 sample indexes available for P5 and P7 Illumina index primer sequences. IDT scientists have tested and validated these 96 sample index sequences to ensure optimal performance.

  • Maximize unique sample identification opportunities with up to 9216 combinations
  • Select only the sequences you need for either unique dual indexing or combinatorial indexing
  • Easily add index sequences to amplicons during rhAmpSeq library preparation using rhAmpSeq Index Primers and the rhAmpSeq Library Kit


rhAmpSeq Index Primers

Add unique "barcode" sequences and P5/P7 sequences to your amplicon libraries. Priced per primer.

To complete your rhAmpSeq workflow, you will also need a rhAmpSeq primer panel—either Custom rhAmpSeq Panel or the predesigned rhAmpSeq Sample ID Panel—and the rhAmpSeq Library Kit.

rhAmpSeq Index Primers are used in the second amplification step of the rhAmpSeq workflow, Indexing PCR 2, to add both unique index sequences and the P5/P7 sequences recognized by Illumina sequencing instruments (Figure 1). 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, Targeted rhAmp PCR 1, creates dual-indexed libraries.

IDT scientists designed and validated the 96 index sequences for optimum performance.

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.

Technical details

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. Each protocol requires a different amount of index primer per reaction, as shown in Table 1.

Table 1. Amounts of index primer needed.

Protocol1X concentration of primer per reactionNumber of reactions per 6 nmol of primer
Regular500 nM600
High-throughput100 nM3000

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.

ConsiderationsRegular protocolHigh-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 hr1–1.5 hr
Total workflow time*4–6 hr4–4.5 hr
* Estimated time to process 12–96 samples using manual pipetting, including reaction setup, cleanup, library quantification, and normalization steps