Input Requirements

Input Requirements

The TruSight Enrichment DNA Sample Prep Kit protocol is optimized for 50 ng of genomic DNA total. Illumina strongly recommends quantifying the starting genomic material.

Input DNA Quantitation

The TruSight Enrichment DNA Sample Prep Kit uses an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of enrichment strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

To obtain an accurate quantification of the DNA library, it is recommended to quantify the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods) should be avoided because common contaminants such as ssDNA, RNA and oligos are not substrates for the TruSight Enrichment assay.

Assessing DNA Quality

Absorbance measurements at 260 nm are commonly used to quantify DNA. The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8-2.0.