DNA Input Recommendations
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The Illumina DNA Prep with Enrichment protocol is compatible with high-quality, double-stranded genomic DNA (gDNA) inputs of 10–1000 ng. For human gDNA samples and other large complex genomes, the recommended minimum gDNA input is 50 ng.
The Illumina DNA Prep with Enrichment workflow is compatible with blood, saliva, or FFPE samples when using the following protocols and reagent kits:
| • | Illumina Blood Lysis Protocol (blood) with the Flex Lysis Reagent Kit |
| • | Illumina Saliva Lysis Protocol (saliva) |
| • | QIAGEN AllPrep DNA/RNA FFPE Kit for extraction of FFPE samples (FFPE) |
| • | Infinium FFPE QC Kit for qualification (FFPE) |
The recommended number of PCR cycles for the EBLT PCR program is adjusted based on sample input concentration and quality. For more information, refer to Amplify Tagmented DNA.
|
Sample Input Type |
Quantification of Input DNA Required |
Required DNA Input Quality |
Normalized Pre‑Enriched Library Yield |
|---|---|---|---|
|
10–49 ng genomic DNA |
Yes |
A260/280 ratio of 1.8–2.0 and A260/230 ratio of 2.0–2.2 |
No |
|
50–1000 ng genomic DNA |
No |
A260/280 ratio of 1.8–2.0 and A260/230 ratio of 2.0–2.2 |
Yes |
|
50–1000 ng extracted FFPE |
Yes |
∆ Cq value ≤ 5 |
No |
|
Saliva |
No |
Not applicable |
Yes |
|
Blood |
No |
Not applicable |
Yes |
Assess gDNA Purity
Use one or more of the following strategies to assess gDNA purity, and make sure that the initial gDNA sample does not contain any organic contaminants, such as phenol and ethanol.
The input DNA must also contain less than 1 mM EDTA. These substances can interfere with the tagmentation reaction and result in assay failure.
| • | UV absorbance is a common method used for assessing the purity of a gDNA sample. The ratio of absorbance at 260 nm to 280 nm provides an indication of sample purity. This protocol is optimized for gDNA with A260/280 ratios of 1.8–2.0, which indicates a gDNA sample with high purity. |
| • | For a secondary indication of sample purity, use an A260/230 ratio. Target an A260/230 ratio of 2.0–2.2. Values outside this range indicate the presence of contaminants. For a complete list of contaminants, including sources, avoidance, and effects on the library preparation, refer to the Illumina support site. |
| • | Dilute the starting material in 10 mM Tris-HCl, pH 7.5–8.5. Incomplete tagmentation caused by contaminants can cause library preparation failure, poor clustering, or low quality sequencing results. |
Enrichment Panel Recommendations
Supplemental enrichment panels can be designed to add or boost coverage of Illumina Custom Enrichment v2 panels. (Supplemental enrichment panels for Illumina Custom Enrichment Panels are not supported for Illumina DNA Prep with Enrichment.)
The Illumina DNA Prep with Enrichment workflow is compatible with the following fixed and customizable biotinylated oligo enrichment panels:
| • | TruSight One – Enrichment Oligos |
| • | TruSight One Expanded – Enrichment Oligos |
| • | TruSight Hereditary Cancer – Enrichment Oligos |
| • | Illumina Exome Panel (CEX) |
| • | Illumina Custom Enrichment Panel v2 |
| • | Illumina Custom Enrichment Panel |
DesignStudio Assay Design Tool supports assay designs for human hg19 and hg38 genome assemblies. Non-human custom enrichment panel designs are supported through Illumina Technical Support. The Illumina DNA Prep with Enrichment workflow is compatible with the following assays:
| • | Illumina Custom Enrichment Panel v2 |
| • | Illumina Custom Enrichment Panel |
If using third-party biotinylated DNA probes (fixed or custom panels), make sure they meet the requirements in Third-Party Panel Requirements.
