DNA Input Recommendations

The Illumina DNA Prep with Exome 2.5 Enrichment protocol, including optional mitochondrial DNA Enrichment is compatible with high-quality, double-stranded human genomic DNA (gDNA) inputs of 10–1000 ng. The recommended minimum gDNA input is 50 ng for optimal performance given the complexity and size of the human genome.

Assess gDNA purity to 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 or poor results.

gDNA Input ≥ 50 ng

For gDNA inputs between 50–1000 ng, quantifying and normalizing the initial gDNA sample is not required. The pre-enrichment library yield (before pooling and enrichment) is automatically normalized during library prep.

gDNA Input < 50 ng

This protocol does not normalize final pre-enrichment library yields when input gDNA ranges from 10–49 ng. Therefore, quantification and normalization of libraries before and after enrichment is required.

If using 10–49 ng gDNA input, quantifying the initial gDNA sample to determine the number of PCR cycles required for the pre-enrichment PCR is recommended. Use a fluorometric-based method to quantify double-stranded gDNA input. Avoid methods that measure total nucleic acid, such as NanoDrop or other UV absorbance methods.

Assess gDNA Purity

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 that may impact the tagmentation reaction. Incomplete tagmentation caused by contaminants can lead to library preparation failure, poor clustering, or low quality sequencing results.

These contaminants include general inhibitors of enzymatic reactions such as:

•

Proteins that coat/bind DNA, preventing library prep enzymes from binding to the DNA substrate.

•

Chelators such as EDTA, salts, and polysaccharides that bind-required cofactors of the library prep enzymes.

•

Other enzymes such as proteases and reagents such as detergents and phenol that degrade or unfold the library prep enzymes.

If this testing produces ratios outside of the acceptable limits, one option is to repurify the gDNA sample using methods with Illumina Purification Beads outlined in Single IPB Methodology (Optional).