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 IDT Technical 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|