NextSeq 1000/2000 loading optimization for Standard SBS kits
These recommendations are for NextSeq 1000/2000 Standard SBS chemistry kits only. When using NextSeq 1000/2000 XLEAP-SBS chemistry, reference the NextSeq 1000/2000 Loading Optimization for XLEAP-SBS Kits article. When sequencing libraries that have not been optimized on the NextSeq 1000/2000 with Standard 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. The optimal loading concentration depends on the library type and insert size. Final loading concentrations for validated library types can be found in the Illumina Denature and Dilute Guide. Starting Point for Standard SBS Loading concentrations For unlisted library types, start with a loading concentration of 650 pM for onboard denature/dilute, and 65 pM for manual denature/dilute, and optimize this concentration over subsequent runs to identify a loading concentration that consistently yields data that meets specifications.
To optimize loading concentration for high diversity libraries, use the %Loading Concentration metric in the PrimaryAnalysisMetrics.csv output file available after run completion. If the %Loading Concentration is <95%, increase the loading concentration in 100 pM increments for on board denature/dilute and 10 pM increments for manual denature/dilute over subsequent runs.
For more information about finding % Loading Concentration, see the Knowledge Base Article: Understanding Loading and Percent Loading Concentration on the NextSeq 1000/2000
If the %Loading Concentration is less than 95%, increase loading concentration in 100 pM increments until 95% loading concentration is achieved.
\* When manually denaturing and diluting a library, the final loading concentration will be approximately 1/10th of that used for onboard denature/dilute.
For any feedback or questions regarding this article (Illumina Knowledge Article #2435), contact Illumina Technical Support techsupport@illumina.com.
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