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.
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.
|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.
Table 2. Choose the best rhAmpSeq library preparation protocol for your needs.
* Estimated time to process 12–96 samples using manual pipetting, including reaction setup, cleanup, library quantification, and normalization steps
|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|