# 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.
  * **The most critical step of the entire workflow is removing ethanol (EtOH) in the 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.

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