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Old 10-17-2016, 04:17 AM   #1
Location: Europe

Join Date: Oct 2016
Posts: 60
Default High percentage of Off-target reads

we are using TruSight Capture Kits from Illumina for Targeted Sequencing.

Normally we have around 25% off-target reads within one run. But from time to time there are runs with 75-85% off-target reads resulting in low coverage in the target region.

What can be the reason for that?

Our guess is, that it has something to do with the hybridization or the washing step afterwards. Maybe the washing soluting wasn't on room temperature? Maybe the hybridization time was to long (but we never exceeded the 20h recommended in the manual)? If the hybridization time was to long, than in the first or in the second hybridization?

Have you had similiar problems and found out what the reason was? Any other guess?

fin swimmer
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