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Undertagmentation of Nextera XT libraries
Undertagmentation during Nextera XT library preparation causes increased final library sizes. Final libraries above 1.2-1.5 kb average size, including adapters, do not cluster well when sequencing. Larger libraries do not cluster as efficiently as smaller libraries. An ideal Nextera XT library size ranges from 200 bp to 1 kb (though this does depend on the bead ratio used during the post-PCR clean-up step as well).
One cause of undertagmentation is using more than 1 ng of input DNA. The final library size will be dictated by the input DNA to transposome ratio, so it is very important that input is quantified with a fluorometric method specific to double stranded DNA, and that exactly 1 ng of input DNA is used. Depending on the concentration of the original DNA sample, it may be necessary to use serial dilutions to accurately reach 0.2 ng/µl for the input.
Another cause of undertagmentation is enzymatic inhibitors in the input sample. Chelators such as EDTA, salts, and polysaccharides can inhibit the enzymatic activity of the transposons and lead to reduced final yield, failure of library prep, or low or uneven coverage when sequencing data are analyzed bioinformatically.* Several factors can impair the performance of the Nextera enzyme:
- Proteins can coat DNA, preventing enzyme binding to the substrate.
- Sequestration of enzyme cofactors by EDTA can negatively affect enzyme function.
- Proteinases, detergents, and phenol can degrade the enzyme.
- Changes in ionic strength and pH caused by chemicals left over from DNA extraction.
- If the sample has contaminants that might affect downstream enzymatic reactions, additional purification steps or centrifugal filtration/concentration can help remove contaminants.
Best Practices to avoid undertagmentation: * Use careful sample handling with extraction protocols optimized to purify inhibitor-free DNA.
- Illumina recommends UV spectrophotometry for quality assessment (to detect inhibitors in the isolated DNA samples) and fluorometric-based methods like Qubit or PicoGreen for DNA quantitation.
- When eluting or resuspending nucleic acids with water, make sure that the pH is 7.0-8.5.
- In the absence of a buffering agent, store samples at -25°C to -15°C to prevent degradation. Samples can also be stored in 10 mM Tris-HCl pH 8.5 with no EDTA.
- Make single-use aliquots of the input sample to prevent cross-contamination and avoid repeated freeze-thaw cycles.
Last modified 2mo ago