Input recommendations for the Illumina 5 Base WGS and Enrichment kits

What is the cfDNA and gDNA amount range for the library prep? Any low or high limit?

• cfDNA: 1 - 20 ng recommended

• gDNA: 50 - 100 ng recommended

• To clarify: the gDNA assay is designed to start with 50-100 ng of material going into sonication. gDNA that is sheared should average ~450 bp.

What is the input amount required for the targeted sequencing/enrichment workflow?

• The DNA input amount is the same as the non-enrichment workflow. The main difference is the additional PCR cycles.

What happens if more than the recommended input amount is used?

• Too much input saturates the enzyme and results in under-conversion.

Is it possible to run low input gDNA samples, < 50 ng?

• The Assay is optimized for 50 - 100 ng inputs. Lower inputs may reduce library complexity and thus impact single nucleotide variant (SNV) and Indel recall and precision. Illumina can recommend the following as a starting point for optimization. Labs will need to optimize and validate the use of low input in this assay:

  Section	Modification
  Prepare Sheared gDNA	Dilute GDMC 1:10 before adding 2.5 µl to gDNA for shearing
  Ligate Adapters	Add 2 µl of UMI3 adapters instead of 5 µl
  Clean Up Ligation	Perform a second consecutive 0.8x bead clean up after ligation
  Index PCR	Run PCR for 9 cycles, instead of 6 cycles
  Clean Up Index PCR	Add 70 µl of IPB instead of 90 µl

• The diluent for GDMC is RSB which is provided in ample excess volume in the kit.

How should the input be quantified?

• gDNA: Illumina generally recommends fluorometric methods of quantification, such as Qubit dsDNA Broad Range Assay Kit for gDNA input quantification (catalog# Q32851 or Q32854). gDNA concentration can also be assessed by TapeStation using the Genomic DNA ScreenTape, although gDNA quality information is not required.

• cfDNA: quantify only the mononucleosomal peak (~ 75 bp to 250 bp) using a size-based quantification method such as the Agilent Fragment Analyzer or Agilent TapeStation. Details are in the library prep Product Documentation, "DNA Input Recommendations" section.

Which samples types are expected to be compatible with the Illumina 5-base solution?

• The assay is compatible with multiple sample types, including cell-free DNA, and DNA extracted from blood, cell lines, or fresh-frozen tissue.

Is it possible to start with degraded or FFPE DNA input and skip/reduce fragmentation**?**

• Illumina recommends the following for FFPE or degraded input. Labs may need to optimize the use of FFPE or degraded input in this assay.

• Follow the “Prepare Sheared gDNA” section but with the following modifications:

1. The recommended input of extracted FFPE DNA is 40 ng. The protocol accepts 1, 20, or 25 ng, as well. Quantification is based on Qubit High Sensitivity dsDNA assay. Only samples with delta Cq < 5 sample quality by the TruSight FFPE QC kit have been tested.

• For each sample, combine 1, 10, 25, or 40 ng FFPE DNA in a volume of 17.5 µl, or volume suitable to your sonicator, plus 2.5 µl of diluted or stock GDMC in the sonication tube or well. For inputs < 40 ng, dilute GDMC 1:10 with RSB prior to adding to sonication tube or well. If the volume is less than the recommended volume for the sonication equipment, add RSB accordingly. The total volume must be less than 50 µl.

1. Sonicate samples to ~200 bp. Transfer sheared material to the corresponding well of the SS1 plate.

• Illumina tested shearing conditions:

Instrument	LE220 Plus	R230
Volume (µl)	20	20
Peak incident power	260 watts	280 watts
Duty Factor	26%	25%
Cycles per burst	50	50
Temperature	10C	10C
Dithering	1 mm Y at 20 mm/sec	3 mm Y at 20 mm/sec
Repeat Process Duration	10 sec	10 sec
Number of Iterations	11	11
Delay Time	10 sec	10 sec
Total treatment time	110 sec	110 sec

1. Bring the volume up to 50 µl with RSB (~32 µl of RSB for 20 µl shearing reaction).

2. Omit the "Prepare Size Select Sheared gDNA" and go directly to "Perform End Repair" using 45 µl of the sheared gDNA.

3. In "Ligate Adapters," add 5 µl of LIGX and 5 µl of diluted UMI3. Dilute UMI3 with RSB based on the following table first:

FFPE Input into shearing (ng)	Volume UMI3 (µl)	Volume RSB (µl)
1	1.12	18.88
10	1	4
25	2	3
40	5	0

1. At "Index PCR," increase number of PCR cycles based on the following table:

Input (ng)	Cycles
1	12
10	10
25	9
40	8

1. In "Clean Up Index PCR," add 80 µl of IPB to each sample

• Typical yield for 1-40 ng of FFPE input is 4 to 30 ng/µl

• Note: these instructions were last updated May 2026

• The flyer titled A single assay to simultaneously detect somatic variants and methylation signatures in FFPE samples presents data generated from FFPE samples.

Which species of input are supported?

• All species are compatible with the Illumina 5-Base DNA Prep kit.

• For the Illumina 5-Base DNA Prep with Enrichment kit, only human (hg19, hg38) and mouse (mm10, mm39) custom panel designs are supported in DesignStudio. For other species or other features not available through DesignStudio, please contact your Illumina Sales Representative to connect you with Illumina’s Concierge Design team.

Which blood collection tubes are supported for cfDNA input?

• Apostle MiniMax Cell-Free DNA Blood Collection Tube (BCT)

• Qiagen PAXgene Blood ccfDNA Tubes

• Streck Cell-Free DNA BCT

• BD Vacutainer Plastic Blood Collection Tubes with K2EDTA

Does Illumina have recommendations for gDNA or cfDNA isolation kits?

gDNA:

Illumina has tested but not fully validated the following kits; each lab must validate performance in their lab. Alternative extraction kits are also acceptable. Illumina recommends DNA extraction methods leveraging silica membranes for purification.

• Whole Blood: QIAwave DNA Blood & Tissue Kit (Qiagen, Hilden, Germany; part number 69554)

• Fresh frozen tissue: QIAamp Fast DNA Tissue Kit (Qiagen, Hilden, Germany; part number 51404)

• Cell-line DNA: NucleoSpin Tissue DNA Kit (Machery-Nagel, Duren, Germany; part number 740952.50)

cfDNA:

Illumina has validated the following kits:

• QIAGEN QIAamp Circulating Nucleic Acid Kit (without carrier RNA)

• QIAGEN QIAamp MinElute ccfDNA Midi Kit

• Zymo MAGicBead cfDNA Isolation Kit

Are there any known inhibitors of the prep?

• Ethanol is a known inhibitor of the conversion step. That is why the workflow has steps to prevent ethanol carryover into the conversion step.

• Illumina does not recommend phenol chloroform extraction, as chemical carryover can negatively impact performance. The phenotype of this type of carryover is that the methylation conversion rates are highly variable from sample-to-sample with no apparent pattern.

• K2EDTA tubes have been validated and should behave like Streck tubes if the workflow is properly executed.

• It is possible that some library prep inhibitors are present in blood. The most common exogenous/endogenous interfering substances (EDTA, Acetaminophen, Triglycerides, creatinine) have been tested on an the cfDNA workflow/chemistry and did not impact the library yields or conversion metrics.

For the initial sonication step, can labs use DPSB (Dulbecco's Phosphate-Buffered Saline) to dilute samples for shearing, or should the Resuspension Buffer (RSB) provided by Illumina be used?

• Illumina recommends shearing in RSB.

Can samples that are a mix of human and mouse DNA be run with this prep?

• In theory, as long as the DNA is "clean" (eg, some silica column is used as part of the DNA purification) we would expect the methylation conversion to work. Analysis-wise, the customer will need to set up a custom genome of both human and mouse, will need to sort reads during alignment to the correct genome, and be aware of some amount of mapping ambiguity for any reads with high human/mouse sequence identity.

• The user may need to make a custom reference via the DRAGEN Reference Builder.

For any feedback or questions regarding this article (Illumina Knowledge Article #9945), contact Illumina Technical Support [e-mail].