Sample Input for Illumina DNA Prep, (M) Tagmentation Library Preparation Kit
**How should the quality of input DNA be assessed?*Assess the quality of genomic DNA (gDNA) by running an aliquot of the sample (approximately 10-100 ng) on a 1% agarose gel stained with SYBR Stain. High quality, intact genomic DNA appears as a high molecular weight band (> 10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can indicate the presence of RNA or degraded DNA. Input DNA should have a 260/280 ratio of ~1.8 and a 260/230 ratio of 2.0-2.2. Additionally, input DNA should not contain EDTA (1 mM upper limit, though no EDTA is best), SDS, phenol, ethanol, or other inhibiting agents such as: Proteins that can coat DNA and prevent enzyme binding to the substrate.
Agents that can sequester enzyme cofactors can negatively affect enzyme function.
Proteinases, detergents, and phenol can degrade enzymes.
Any chemicals left over from DNA extraction that can alter the ionic strength or pH of DNA storage buffer, which might adversely affect the tagmentation reaction.
Which method is recommended to quantify DNA for the Illumina DNA Prep, (M) Tagmentation protocol?* For DNA inputs between 100-500 ng, accurate quantification of the initial DNA sample is not required, and normalization of the final yield is expected.
Quantification is recommended if using less than 100 ng DNA input. To quantify input DNA, use a fluorometric-based method that is specific to double-stranded DNA.
The concentration of gDNA can be determined using the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and can detect samples in a concentration range from 10 pg/μl -1000 ng/ μl.
For more information, see the DNA Input Recommendations section of the Illumina DNA Prep, (M) Tagmentation Reference Guide.
**Is the substrate for tagmentation exclusively dsDNA? Is ssDNA or RNA compatible?*Illumina DNA Prep is optimized for gDNA (dsDNA) and will not work on ssDNA or RNA. How much input genomic DNA is required for the Illumina DNA Prep, (M) Tagmentation workflow? The protocol is compatible with DNA input ranging from 1-500 ng.
For human DNA samples and other large complex genomes, the recommended DNA input is between 100-500ng.
For small genomes, the DNA input amount can be reduced to as low as 1 ng. The number of PCR cycles should be modified in accordance with the protocol.
**What is the minimum amount of DNA compatible with the built-in library normalization?**≥ 100 ng. **What happens if less than 100 ng DNA input is used for a human sample?**Library yield is not normalized. This could result in reduced diversity and increased duplicates. **What happens if more than 500 ng DNA input is used?**Although testers have successfully generated libraries from more than 500 ng input DNA, this application is not supported.
Has Illumina tested GC-rich or AT-rich genomes? Is there any bias? * A variety of genomes have been tested and the data remains consistent.
Demo datasets for Illumina DNA Prep libraries prepared with a variety of genomes from bacteria, plants, agriculture, and Humans can be found on BaseSpace.
**Are degraded or FFPE samples compatible with this protocol?**Illumina does not support FFPE or degraded DNA as input for this protocol. The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using this protocol. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina. Can PCR amplicons be used as input? Yes. The PCR amplicon must be > 150 bp. Shorter amplicons can be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.
For any feedback or questions regarding this article (Illumina Knowledge Article #1262), contact Illumina Technical Support techsupport@illumina.com.
Last updated