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.

For any feedback or questions regarding this article (Illumina Knowledge Article #1054), contact Illumina Technical Support [e-mail].