NextSeq 1000/2000 Loading Optimization for XLEAP SBS kits
These recommendations are for NextSeq 1000/2000 XLEAP-SBS chemistry kits only. When using NextSeq 1000/2000 Standard SBS chemistry, reference NextSeq 1000/2000 Loading Optimization for Standard SBS kits.
When sequencing libraries that have not been optimized on the NextSeq 1000/2000 with XLEAP-SBS chemistry, it is strongly recommended to perform titration runs. Even if a loading concentration is provided in the user guide, the loading concentration may need optimization.
Starting Point for XLEAP-SBS Loading concentrations
For library types that are not within our user guide, the starting XLEAP-SBS loading concentration for your titration will vary by flow cell. We recommend:
For P1/P2, start with the on-market/standard SBS loading concentration for the application, or loading concentration previously titrated for standard SBS.
For P3/P4 and 600-cycle P1/P2, reduce the standard SBS on-market loading concentration for the application by approximately 25%.
eg, if the standard SBS recommendation is 650 pM, start with 488 pM for XLEAP-SBS
And then use the primary and secondary sequencing metrics listed later in this article to optimize the loading concentration*.*
Additional development work is occurring, and loading concentration recommendations are being added to Illumina Product Documentation in the future. Check with the local Field Application Scientist (FAS) or technical support to see if there development-tested recommendations for the application of interest.
XLEAP-SBS loading concentrations may be different from loading concentration on the same instrument than when using standard SBS. Unlike for standard SBS, the loading concentration of the same library may be different for different kits (ie, different flow cell types and long-read vs short-read kits). Titrations should be performed separately for each kit configuration.
XLEAP Loading Concentration Titration Guidance
The optimal loading concentration range for each lab may be different depending on what metrics matter to each user. Evaluating primary metrics of output reads, %Loading Concentration, and %PF, as well as secondary metrics including duplicates and coverage is strongly recommended when determining the optimal loading concentration.
For more information about finding % Loading Concentration, please see the Knowledge Base Article: Understanding Loading and Percent Loading Concentration on the NextSeq 1000/2000
Size-normalized qPCR is the quantitative method used internally by Illumina. Using a different quantitative method to determine library stock concentration may affect the titration process and the optimal loading concentration.
For titrating libraries, center titrations around +/- ~25% of the recommended loading concentration
For example, if using a library type that has a targeted starting concentration of 1000 pM
75%: use 750 pM
125%: use 1250 pM
Typical Metric Trends for Loading Concentration Titrations
This figure shows how the different parameters change with input concentration (disclaimer: onboard Denature & Dilute was used). In this case, optimum loading concentration is approx. 99.5%.
Example(s) of how to apply the metrics:
A sequencing run with high %PF, output reads, and high duplicates is an indication of underloading - To resolve this issue, increase the loading concentration.
A sequencing run with very high occupancy, low % PF and low duplicates is an indication of overloading - To resolve this issue, decrease the loading concentration.
Usually Proper Loading reached at “% Loading Concentration” of 95% - 99%
Titration Example: Illumina DNA Prep with Enrichment Exome on XLEAP P4
Note: the optimal loading concentration may vary when this library prep is used with different panels.
In this case optimum loading concentration is approx. 97% (disclaimer: onboard Denature & Dilute was used), which is the Product Documentation Guidance for Illumina DNA Prep with Enrichment.
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