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

  • Note: 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.

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?

FFPE or degraded inputs are not officially supported sample types, however Illumina can recommend the following as starting points for optimization. Labs will need to optimize and validate the use of FFPE or degraded input in this assay:

• Use input recommendations for gDNA of 50 ng - 100 ng input.

• Adjust shearing conditions to those described for Illumina FFPE DNA Prep with Exome 2.5 Enrichment Prep. A maximum volume for sonication of 50 µl is required for compatibility with the End Repair step.

• Omit the "Size Select Sheared DNA" step, as that step is intended to remove smaller fragments, that should be retained when processing FFPE.

• Include two rounds of IPB cleanup in the "Clean Up Ligation" step.

• Increase number of PCR cycles to up to 10 cycles.

Which species of input are supported? If not all species, is this a library prep or an analysis limitation?

• Human and mouse are the only species supported. This is an analysis limitation.

• For the Enrichment kit, only human (hg 19, hg38) and mouse (mm10, mm39) are supported for custom panel designs in DesignStudio.

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.

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