Pool Pre-Enrichment Libraries

This step combines DNA libraries with unique indexes into one pool of up to 12 libraries. Fewer pre-enrichment libraries may be pooled, but you may need to perform additional optimization. If using fewer pre-enrichment libraries, you cannot process the full 96 samples through enrichment, as only eight enrichment reactions are supported with this kit.

You can pool by volume or mass. Use the following table to determine the appropriate method for your input.

Recommended Pooling Methods
Sample Input	Pooling Method
10–49 ng gDNA	Mass only
50–1000 ng gDNA	Mass or volume*
Saliva	Volume
Blood	Volume

* If starting with ≥ 50 ng DNA input, the pre-enrichment library yields were normalized during tagmentation, which uses eBLT. This normalization enables you to pool equal volumes of each pre-enrichment library in a final pool volume ≤ 30 µl (target 250–500 ng per sample).

After pre-enrichment library quantification, all sample input types can be pooled by mass to achieve optimal library balance and a similar number of sequencing reads per library.

The final yield of pre-enrichment libraries generated in separate experimental preparations can vary. Therefore, pooling by mass is recommended to achieve optimal library balance when pooling samples from multiple experimental preparations.

If pre-enrichment libraries are not quantified, then proceed to pooling by volume. Pooling by volume can only be performed when starting with ≥ 50 ng gDNA input.

Quantify Pre-Enrichment Libraries

Determine library concentration (ng/µl) by proceeding as follows:

Quantify 1 μl of each pre-enrichment library using the Qubit dsDNA BR Assay Kit to determine library concentration (ng/μl).

Expect the following pre-enrichment library yield based on sample type and input.

Sample Input Type (ng)	Pre-enrichment Library Yield (ng)
10–49 gDNA	≥ 100
50–1000 gDNA, blood, saliva	≥ 500

Concentration results may differ depending on the quantification method used. The Qubit dsDNA BR Assay is recommended, but validation will be needed when using an alternative method.

Pool by Volume

When the input is 50–1000 ng gDNA, quantifying and normalizing individual libraries generated in the same experiment is not required.

To achieve optimal performance, only pool pre-enrichment library samples prepared by the same user, reagent lot, and index adapter plate.

If starting from 50–1000 ng gDNA input, saliva input, or blood input, you can pool the pre-enrichment libraries using the following standard protocol.

For a standard 12-plex pool: Combine 2.5 μl of each pre-enrichment library in a 1.7 ml microcentrifuge tube to generate a 12-plex pool at a total final pool volume of 30 μl.

When preparing a pool of lower plexity (<12 pre-enrichment libraries per pool): Combine 2.5 μl of each pre-enrichment library in a 1.7 ml microcentrifuge tube, then add RSB to bring the total final pool volume up to 30 μl.

Using the sample tracking method you chose in Prepare for Pooling, record the indexes for the libraries you plan to pool in this step.

Pool pre-enrichment libraries based on the sample volumes in the following table.

Library Pool Plexity	Each Pre-Enrichment Library Volume (µl)	Total Volume (µl)
12-plex	2.5	30

SAFE STOPPING POINT

If you are stopping, cap the 1.5 ml microcentrifuge tube and store at ‑25°C to ‑15°C for up to 30 days.

Pool by Mass

Using the sample tracking method you chose in Prepare for Pooling, record the indexes for the libraries you plan to pool in this step.

Combine each library in a 1.7 ml microcentrifuge tube to generate a 12-plex pool shown in the following table. Repeat as needed for additional pools.

When pooling by mass, always pool equivalent masses of each pre-enrichment library to obtain similar sequencing read output from each final, enriched library.

If the volume of your 12 pooled libraries is > 30 µl, concentrate the pooled libraries to 30 µl. Refer to Concentrate Pooled Libraries (Optional).

If the final pool volume is less than 30 µl, add RSB to bring the total final pool volume up to 30 µl.

Pooling by Mass Guidelines
	Inputs per Pre-enrichment Library (ng)			Total Mass per Final 12-Plex Pool (ng)
Sample Input	Minimum	Maximum	Recommended	Minimum	Maximum	Recommended
10-49 ng gDNA	100	500	250–500	1200	6000	6000
[Optional] 50–1000 ng gDNA*	250	500	250–500	3000	6000	3000–6000
[Optional] Quantified saliva and blood gDNA*	250	500	250–500	3000	6000	3000–6000

*If starting from 50–1000 ng input, saliva input, or blood input, you can pool pre-enrichment libraries by volume instead of by mass. Pooling by mass will produce more consistent sequencing reads from each enriched library within the pool.