Nextera Rapid Capture Enrichment 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) such as QuantiFluor and running samples in triplicate to obtain more confident measurements. 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 Nextera Rapid Capture Enrichment 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.
The Nextera Rapid Capture Enrichment protocol has been optimized for 50 ng of total gDNA. A higher mass input of gDNA can result in incomplete tagmentation and larger insert sizes, which can affect enrichment performance. A lower mass input of gDNA or low quality gDNA in the tagmentation reaction can generate smaller than expected insert sizes. Smaller inserts can be lost during subsequent cleanup steps and result 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/ul. Samples are then requantified using a similar fluorometric-based method and normalized to a final 5 ng/ul.