Input Requirements

TruSight Cardio library preparation 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. Therefore, accurate quantitation of the gDNA is essential.

Illumina recommends quantifying the starting gDNA using a fluorometric-based method specific for double-stranded DNA (dsDNA) and running samples in triplicate to obtain more confident measurements. 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 Cardio Sequencing Panel.

  • Avoid methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods)
  • Common contaminants, such as ssDNA, RNA, and oligos, are not substrates for the TruSight Cardio Sequencing Panel assay
  • Make sure that the starting DNA does not contain more than 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. For more information, see DNA Quantitation on page 1 of the TruSight Cardio Library Preparation Guide.
  • DNA samples can contain substances that interfere with the Nextera tagmentation reaction and result in unexpected library insert sizes. Therefore, Illumina recommends an optional sample cleanup, followed by requantifying the DNA samples, before beginning library preparation. The additional cleanup makes sure that the conditions are optimal for TruSight Cardio Sequencing Panel library preparation.

The TruSight Cardio protocol has been optimized for 50 ng of total gDNA. A higher mass input of gDNA may result in incomplete tagmentation and larger insert sizes, which can impact enrichment performance. Conversely, a low mass input of gDNA or low quality gDNA in the tagmentation reaction may generate smaller than expected insert sizes, which can be lost during subsequent clean-up steps resulting in lower diversity.

To minimize gDNA sample input variability into the tagmentation step, Illumina strongly recommends a two-step method of gDNA normalization. After the initial quantification, gDNA samples are first normalized to 10 ng/μl. Samples are then re-quantified using a similar fluorometric-based method and normalized to a final 5 ng/ul.