I'm new to the library prep side of sequencing and I've been having trouble getting my post-tagmentation, post-amplification libraries to match the profile in the Nextera DNA Exome protocol.
I've repeated the process a few times with increasing care (using low-bind tubes, checking input concentrations with Qubit, testing beads on a DNA ladder, etc.) and I'm finding that I have a flat, right skewed bioanalyzer peak (the peak should be between 200-500bps and mine hangs over 1000bps - see attached trace). Illumina's FAQ claims that a secondary, larger peak is common and should be of no concern since it's often an amplification product but I'm not convinced that's what I'm seeing here.
Has anyone else had a pre-enrichment library prep with a similar profile? Is this cause for concern (over- or under-tagmentation)? If so, do you have any tips on how the protocol can be adjusted to account for it?
I've repeated the process a few times with increasing care (using low-bind tubes, checking input concentrations with Qubit, testing beads on a DNA ladder, etc.) and I'm finding that I have a flat, right skewed bioanalyzer peak (the peak should be between 200-500bps and mine hangs over 1000bps - see attached trace). Illumina's FAQ claims that a secondary, larger peak is common and should be of no concern since it's often an amplification product but I'm not convinced that's what I'm seeing here.
Has anyone else had a pre-enrichment library prep with a similar profile? Is this cause for concern (over- or under-tagmentation)? If so, do you have any tips on how the protocol can be adjusted to account for it?
Comment