What causes variation of read counts (CV > 10%) among libraries within Normalase™ pools?

There are several causes for this which may include:

• Libraries not meeting the 6 nM or 12 nM minimum threshold in 20 µL library yield
• Sequencer overloading and subsequent over-clustering resulting in low sequencing quality
• Inconsistent pipetting of Normalase™ I Master Mix into individual libraries
• Inconsistent pipetting of individual libraries into pools before Normalase II

Each of these conditions could introduce more variability and increase variation of read counts to >10%.

Check that your libraries meet the minimum threshold of 6 nM or 12 nM in a 20 µL library yield for optimal performance.

Note: Quantify problematic libraries before pooling to confirm suggested molarity. Use a P10 pipette if available, and ensure pipetting equipment is maintained and calibrated.