Optimizing coverage for targeted resequencing

Determining the distribution of coverage depth for targeted regions requires the generation of normalized coverage plots. Simply calculating the mean sequencing coverage will provide only a summary of the average read depth across the bases targeted in the enriched sample. The most commonly used methods report a given percentage of targeted bases covered at a particular depth (eg, 90% of targeted bases covered at 10× read depth). It is possible to increase the total sequencing coverage by deeper sequencing.

TruSeq exome analysis scripts generate mean normalized coverage plots, showing the distribution of coverage depth across all targeted bases. This enables researchers to calculate just how much sequencing will be required to yield a given percentage of targeted bases at a particular read depth. It is important to note that this normalization is a characteristic property of the Illumina TruSeq Exome Enrichment assay, averaged over a number of runs. TruSeq analysis scripts have been designed to report the distribution of depth of coverage in both an absolute and normalized manner (eg, 90% of targeted bases covered at 0.2× of the normalized mean).

Information regarding Creation of Cumulative Normalized Coverage Plots and How to Calculate the Required Amount of Sequencing Data can be found in the Technical Note Optimizing Coverage for Targeted Resequencing.

• The information outlined in this technical note can also be helpful for calculating and optimizing target coverage for other targeted sequencing panels.

For any feedback or questions regarding this article (Illumina Knowledge Article #1409), contact Illumina Technical Support [e-mail].