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 reference guide, the loading concentrations may need optimization.

Starting Point for XLEAP-SBS Loading Concentrations

• For library types that do not have starting loading concentration guidance, the starting XLEAP-SBS loading concentration for titration will vary by flow cell. Illumina recommends:

  • eg, if the standard SBS recommendation is 650 pM, start with 488 pM for XLEAP-SBS.

• For P1/P2, start with the standard SBS loading concentration for the application, or loading concentration previously titrated for Standard SBS kits.

• For P3/P4 and 600-cycle P1/P2, reduce the standard standard SBS loading concentration for the application by approximately 25%.

• Optimize the loading concentration using the primary and secondary sequencing metrics listed later in this article.

XLEAP-SBS loading concentrations may be different from loading concentration on the same instrument when using standard SBS. Unlike 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 which metrics matter more 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, see 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 quantification 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 (Figure 1)

Figure 1. This figure shows how the different parameters change with input concentration (disclaimer: onboard Denature & Dilute was used). In this case, optimum loading concentration is approximately 99.5%.

Example(s) of how to apply the metrics:

• A sequencing run with higher-than-expected PhiX alignment, low %PF, occupancy, output reads, and high duplicates is an indication of underloading - To resolve this issue, increase the loading concentration.

• A sequencing run with lower-than-expected PhiX alignment, high occupancy (~98-99%), low % PF, and low duplicates is an indication of overloading - To resolve this issue, decrease the loading concentration.

• Usually proper loading is reached at “% Loading Concentration” of 95% - 98%

Titration Example: Illumina DNA Prep with Enrichment Exome on XLEAP P4 (Figure 2)

Figure 2. In this example, optimum loading concentration is approximately 97% (onboard Denature and Dilute was used), which is the Product Documentation Guidance for Illumina DNA Prep with Enrichment. Note:The optimal loading concentration may vary when this library prep is used with different panels.

For any feedback or questions regarding this article (Illumina Knowledge Article #8693), contact Illumina Technical Support [e-mail].