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Does anyone do this with the TruSeq kits? Illumina advertised this feature very strongly in their presentations in my area, as a huge time saving feature of the TruSeq kits as compared to the older sample prep kits... yet the product manual doesn't say anything about it. I'm a bit worried about uneven amplification of the libraries during a pooled PCR. Any comments?
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Would that also cause jumping errors and waste of sequencing reads? -
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I'd like to refresh this thread. Has anyone had success with TruSeq library prep, if you pool your adapter ligated samples prior to the PCR enrichment step?
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Pooling prior to PCR amplification is not recommended. It precludes the option of adjusting the concentrations of individual libraries to obtain equal read depth.Comment
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Done once. DO NOT attempt it. Some libraries just dissapeared.Comment
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We didn't do that. In theory, it may cause unbalanced library pool. Some libraries could have very low sequencing reads.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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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...
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