General workflow FAQ for the Illumina 5 Base WGS and Enrichment kits

Is there any recommendation if the cfDNA size-selection quantification is less than expected (0.2 ng/µl with 10 ng input)?

• A yield of 0.2 ng/µl is expected from an input of 10 ng cfDNA, using the mononucleosomal peak as a reference. Note the assay supports starting with as little as 1 ng mononucleosomal cfDNA. Proceeding with this protocol is recommended is an operator has < 0.2 ng/µl. However, if yields below 0.2 ng/µl are frequently observed when starting with 10 ng mononucleosomal cfDNA, it is advised to review and ensure proper technique during size selection of the mononucleosomal peak.

Can IME also be mixed with pipette mixing? What happens if the MCB + IME master mix tube is vortexed? (The recommendation in the protocol is not to vortex.)

• Pipette mixing and inversion are both okay. The key is that the pipette must be set at close to the full volume of the reagent to ensure proper mixing. Do not vortex IME.Vortexing can lead to lower conversion activity or uneven activity across samples.

Will a dry time of 15 minutes lead to overdried pellet and low elution efficiency?

• No.

The WGS protocol indicates 6 PCR cycles for the Index PCR. Should the the number of PCR cycles be increased for low input?

• Illumina recommends keeping 6 cycles of PCR for all sample inputs (50-100 ng of gDNA or 1-20 ng of cfDNA).

What are the critical steps in the workflow or common mistakes seen?

• Adding more than the recommended amount of sample, which can overwhelm the proprietary enzyme during the conversion step and reduce conversion efficiency. In this case, more is not better.

• Not removing all residual ethanol (EtOH) and beads present following the ligation cleanup step. This can inhibit the IME enzyme in the subsequent conversion step.

  • Most critical step of the entire workflow is removing ethanol (EtOH) in Clean Up Ligation step!

• Not handling IPB beads appropriately.

• Not trimming adapters and UMIs, which leads to high false positive rate of conversion due to the fact that adapters are not methylated. Therefore, if they are not trimmed they contribute to an increase in the False Positive (FP) rate and decreased True Positive (TP) rate.

For any feedback or questions regarding this article (Illumina Knowledge Article #9946), contact Illumina Technical Support [email protected].