Library QC for the Illumina DNA Prep (M) Tagmentation Kit

This FAQ addresses frequently asked questions regarding Illumina DNA Prep library evaluation. See the Reference Guide for more information. Which method is recommended to quantify final libraries prior to sequencing?* When using DNA inputs of 100-500 ng, the normalization features of this library protocol mean that quantification and normalization of individual libraries, generated within a single experiment (batch), are not required.

• Quantify the pool with Qubit or PicoGreen.

• For libraries prepared with less than 100 ng, quantify individual libraries with Qubit or PicoGreen.

How to check the quality of libraries?* Use a Fragment Analyzer (Advanced Analytics) or Bioanalyzer 2100 (Agilent Technologies) to check the quality and intended size distribution of a tagmented sample. For examples of Bioanalyzer traces and library size distributions, see the library prep reference guide. What can cause libraries to look different than the example shown in the Reference Guide?* The profile of the amplified library product can look different from the example depending on the type and quality of input DNA, and the size selection used.

• Inaccurate pipetting during the double sided SPRI will cause fragment size variation. Some variation is expected in the profiles of different libraries.

Is a bump/shoulder/peak at 1000-3000 bp in traces expected?* It is not atypical to see a small bump/shoulder/peak at 1000-3000 bp. The appearance of the peak can vary between DNA samples and does not affect downstream sequencing.

What is the typical size range and average size of final library?* 600 bp average fragment length on Bioanalyzer (150-1500 bp size range).

• 350 bp median insert size from sequencing.

Which equation should be used to calculate the final library concentrations (nM)? See Converting ng/µl to nM when calculating dsDNA library concentration.

For any feedback or questions regarding this article (Illumina Knowledge Article #1258), contact Illumina Technical Support [e-mail].