How do I pool 10x libraries for Illumina sequencing?

Question: How do I pool 10x libraries for sequencing?

Answer: Pooling 10x libraries for sequencing will depend on the library type, target cell number, tissue covered spots (Visium), and desired read depth. Please consult the corresponding User Guide, Sequencing Tech Notes, and the Sequencing Section on the 10x Support Page.

For example:

• Sequencing Requirements for Single Cell 3' 
• Sequencing Requirements for Single Immune Profiling 
• Sequencing Requirements for Visium for fresh-frozen 
• Sequencing Requirements for Visium for FFPE
• Sequencing Requirements for Fixed RNA Profiling

There are product-specific pooling considerations. For example:

• Can Multiome ATAC and Multiome GEX libraries be sequenced together?
• Why can I not pool Multiome ATAC and Standalone ATAC libraries on certain sequencers?
• Can Visium for fresh-frozen libraries be pooled with other 10x libraries?
• Can Visium for FFPE libraries be pooled with other dual indexed 10x libraries?

POOLING

There are many considerations for pooling. We highlight a few important considerations below. It is important to read the product-specific User Guide carefully for each product as well as the knowledge base articles on the Support webpage. We also recommend reaching out to your sequencing provider to see what guidance they might have for library quantification and pooling.

Select unique indexes

To ensure that each library is properly assigned to an input sample, it is important to select different indexes for each library (e.g. do not select the same index for two libraries in a pool). It is also important to consider Illumina guidelines for index selection:

• Is nucleotide diversity in the index read important for Illumina sequencing?

10x Genomics does not support sequencing single index and dual index libraries together in the same lane:

• Can single index and dual index libraries be pooled for sequencing?

Accurately quantify final libraries prior to pooling

Accurate quantification of final libraries is critical for obtaining the desired pool representation. We would recommend qPCR for quantification because it measures the templates that have both adapter sequences, which is a measure of the fragments that will form clusters on a flow cell. These general steps can be followed for accurate library quantification and sizing.

• Follow guidelines for “Post Library Construction QC” outlined in the User Guide.
• Select a region on the fragment analyzer library trace that encompasses the size range of fragments that will cluster. This means fragment size from the library trace will be used as the insert size for further library quantification. 
• Quantify each library by Kapa qPCR quantification as per the instructions in “Post Library Construction Quantification”.
• Please find more information about the Kapa quantification kit here.
• This will yield a concentration (in nM) for each library.
• Size correct the nM obtained by qPCR using the average library size as outlined in the Kapa Quantification User Guide.

Additional details on library quantification and pooling prior to sequencing can be found in Illumina’s Sequencing Library qPCR Quantification Guide.

Putting it all together

 There is a pooling spreadsheet for the Connect which can serve as an example. 

• Average Library Size (bp): Determined by bioanalyzer (or tapestation / LabChip / etc)
• Library Concentration (nM): Determined by qPCR
• Cells per Sample: How many cells you expect for each library (e.g. targeted cell recovery)
• Reads per Cell: This is outlined in each User Guide. 
• This said, different sequencing depths may be necessary depending on the experimental design and cell type(s) of interest. More about Resolving Cell Types as a Function of Read Depth here. 
• Target Volume: can be adjusted up or down to make sure the ‘final transfer volume’ is not too high (e.g. use up too much of final library); or too low (pipette volume too low / inaccurate).
• Target Pool Concentration: This is the expected concentration of the final pool. We highly recommend requantifying the pool prior to sequencing. 
• If the expected concentration is significantly different from the observed concentration (by qPCR) this could indicate a problem with pooling or individual library quantification. 

Alternatively, each library can be diluted to the same nM (by qPCR) and pooled in equal molar ratios as recommended by Illumina here. 

Take into account target cell number when pooling and also differences in library size. Smaller libraries will cluster more efficiently than larger libraries and thus their effective concentration is higher.

Pooling verification
Illumina recommends assessing library representation in a pool using iSeq as pooling QC. More information about this here. We will defer to Illumina’s expertise on pooling verification and also final pool quantification prior to sequencing.
Sequencer selection
Select the appropriate sequencer based on the total reads needed for each pool. The 10x Genomics Flowcell Capacity Calculator can assist with sequencer selection.

Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC+GEX, Single Cell ATAC, Visium for fresh-frozen, Visium for FFPE, Fixed RNA Profiling