Single or double sided bead clean ups with Illumina DNA PCR Free.

Why are two rounds of IPB clean ups performed for each sample? This protocol uses two rounds for a double-sided bead clean up. The first round removes the extremely large fragment size DNA from the library, while the second round removes the extremely small fragment size DNA from the library. Tight library insert size allows for better clustering and leads to consistent and high-quality sequencing metrics.

Why is a two-sided size selection used for the standard workflow? Why not keep higher fragment sizes? Read 2 Q30 scores and other performance metrics can be negatively impacted by larger fragments, so these are removed in the standard library prep workflow.

Can a single-sided 1.8x (or more) bead clean up be used instead of the two-sided clean up? * A single-sided clean up leads to a change in the fragment distribution of the final library. Though this is necessary for the low input protocol, some metrics (such as Q30 and insert size) may be reduced. Outside of the low input protocol, Illumina does not recommend performing a single-sided clean up under normal testing conditions.

• For more details, see the app note Tunable insert sizes with Illumina DNA PCR-Free Prep, Tagmentation.

**Why is the size selection different for low DNA input (25 ng-99 ng) and higher DNA input (100 ng-2000 ng)?**Optimal size selection delivers consistent library fragments and the best quality. If no modification were made, at the lowest inputs, the workflow would generate a very low yield and significantly smaller fragment size. To improve yield and fragment size at low inputs, the single sided clean up was implemented. This change has a minimal negative impact on quality and ensures enough DNA can be obtained.

For any feedback or questions regarding this article (Illumina Knowledge Article #3467), contact Illumina Technical Support [e-mail].