Quality Metrics

Two factors can cause cluster density fluctuations in libraries prepared with the Nextera XT DNA Library Prep:

• An average sample size that is too large or too small after tagmentation.
• A final sample concentration that is too low due to a low yield when starting the bead-based normalization step.

To troubleshoot fluctuations in cluster density, consider checking library size and library concentration. For more information, refer to Nextera XT Library Prep: Tips and Troubleshooting (Pub. No. 770-2015-015).

Larger molecules cluster less efficiently than smaller molecules. If the fragment size after tagmentation is larger than expected, low cluster numbers are possible. The inverse is also true. The average expected library size after tagmentation is between 400 bp and 1.2 kb.

Check the library size with a high sensitivity Bioanalyzer trace after the PCR cleanup step. Look for a long low plateau. Alternatively, PCR-amplify the library with qPCR primers and run the product on an agarose gel. The sequence for these primers is available in the Sequencing Library qPCR Quantification Guide (document # 11322363).

• Short libraries indicate too little input DNA—Requantify the input DNA with a fluorometric method. Start with 10%–25% more input DNA. If the library peak is below 400 bp and you want to continue with this library, dilute the library further.
• Long libraries indicate too much input DNA or the presence of inhibitors—Start with less input DNA, make sure that the input DNA is free from inhibitors, and repeat the quantification step.

For more information on library dilution, refer to the denature and dilute libraries guide for your sequencing system.

Bead-based normalization is most efficient when the library yield after amplification is 10–15 nM, or higher. Measure library concentration using high sensitivity dsDNA Qubit after library cleanup, and measure library size with a Bioanalyzer to calculate molarity.

If you are starting with high-quality DNA and see low yield after library cleanup, there are possible issues with IPB cleanup or the amplification step. If results show either condition, confirm proper storage of the PCR master mix at ‑25°C to ‑15°C in a no-frost freezer. Confirm minimal freeze-thaw cycles.