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.
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.
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.
|Protocol||1X concentration of primer per reaction||Number of reactions per 6 nmol of primer|
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.
|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|
For more information, refer to the analysis guidelines in the User guides and protocols section.