# Overtagmentation of Nextera XT libraries

Samples that have been overtagmented by the transposons have short fragments that will be washed away during the size selection step resulting in **reduced library yield**, which can cause **coverage dropouts**.

Causes of overtagmentation include:

* Inaccurate quantification that results in < 1 ng for the tagmentation step
* Use of FFPE or degraded DNA samples
* Use of smaller sized input. FFPE, degraded samples, and small amplicons < 300 bp are not supported.

Additional best practicesto avoid overtagmentation:

* Illumina recommends UV spectrophotometry for quality assessment and fluorometric-based methods like Qubit or PicoGreen for DNA quantification.
* 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.
* If using amplicon input, design amplicons to be > 300 bp. For additional recommendations for using PCR Amplicon as input, see the [Nextera XT Reference Guide](https://support.illumina.com/downloads/nextera_xt_sample_preparation_guide_15031942.html).

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