DNA Input Recommendations
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The Illumina DNA Prep protocol is compatible with DNA inputs of 1–500 ng or higher. For human DNA samples and other large complex genomes, the recommended minimum DNA input is 100–500 ng. For small genomes, such as microbial, the DNA input amount can be reduced to 1 ng. If the DNA input amount is reduced, the PCR cycling conditions must be modified accordingly.
Assess DNA purity to make sure that the initial DNA sample does not contain > 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the tagmentation react ion and result in assay failure.
The Illumina DNA Prep workflow is compatible with blood and saliva samples when using the following protocols and reagent kits:
| • | Illumina Blood Lysis Protocol (requires Flex Lysis Reagent Kit) |
| • | Illumina Dried Blood Spot Extraction (requires Bead-Linked Transposomes) |
| • | Illumina Saliva Lysis Protocol |
The recommended number of PCR cycles for the BLT PCR program is adjusted based on sample input concentration and quality. If reducing the DNA input amount, modify the PCR cycling conditions accordingly. For more information, refer to Amplify Tagmented DNA.
|
Total DNA Input (ng) |
Quantification of Input DNA Recommended |
Normalized Library Yield |
|---|---|---|
|
< 100 |
Yes |
No |
|
100–500 |
No |
Yes |
|
Blood/Saliva |
No |
Yes |
DNA Input < 100 ng
This protocol does not normalize final library yields from < 100 ng DNA input. Therefore, quantification and normalization of libraries before sequencing is required.
If using < 100 ng DNA input, quantifying the initial DNA sample to determine the number of PCR cycles required is recommended. Use a fluorometric-based method to quantify double-stranded DNA input. Avoid methods that measure total nucleic acid, such as NanoDrop or other UV absorbance methods.
DNA Input 100–500 ng
For DNA inputs between 100–500 ng, quantifying and normalizing the initial DNA sample is not required. Normalized samples produce < 20% coefficient of variance on average when pooled in equal volumes for sequencing.
Access DNA Purity
Assess the DNA purity to make sure that the initial DNA sample does not contain any organic contaminants, such as phenol and ethanol, and contains 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 DNA sample. The ratio of absorbance at 260 nm to 280 nm provides an indication of sample purity. This protocol is optimized for DNA with A260/280 ratios of 1.8–2.0, which indicates a DNA sample with high purity. |
| • | For a secondary indication of sample purity, use the ratio of absorbance at 260 nm to absorbance at 230 nm. Target a 260/230 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 Nextera XT Library Prep: Tips and Troubleshooting Technical Note. |
| • | 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. |
