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We are sending ChIP samples for sequencing on a HiSeq 2500. We sent some to a facility a while ago and they used an Illumina kit for library prep. Now we are planning to switch to a new facility for various reasons including cheaper prices and they use a KAPA library prep kit. We want to repeat the original experiment to have n=2. Will there be a problem, or any kind of bias, or anything else we should worry about with one set being prepared with the Kapa kit and the other with the Illumina kit? Also, are these Kapa kits of comparable quality to the Illumina kits?
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If the libraries are constructed using the same adapter stocks, and amplification is done using the same polymerase, the results should be comparable;
Although the % of input DNA converted to adapter-ligated molecules may differ slightly between the kits, this will be rectified in the amp. However, if different enzymes are used for the enrichment, the enzyme which shows the most bias will produce higher duplication rates i.e. you will have more copies (duplicates) of the "easy" fragments, and fewer duplicates of the "difficult" molecules.
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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