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Hi,
is it possible to pool different species (after ligating individual barcodes off course) for RAD seq on Illumina?
is it possible to pool different species (after ligating individual barcodes off course) for RAD seq on Illumina?
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I would say probably (my lab developed RAD-Seq). We almost always ran multiple projects in a lane, with different species in each. I don't think we ever, however, mixed different species in the amplification. Usually we would pool 16-24 samples before shearing, so there was little reason to pool across species if carrying out a project of 48 flies and another of 72 mimulus. We would make 2 pools of 24 flies and 4 pools of 18 plants.
If one project was cut with SbfI and another with PstI, you would get different shearing efficiencies (better shearing for the SbfI project) and would have difficulties getting the reads balanced as you would like, but otherwise there is little reason to suspect pooling would cause problems. Just little reason to do so.Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com -
Thanks! Ok, so if I understand you correctly: it is lttle risk to mess up completely if we pooled two different species (to a total of 48 samples, all cut with the same enzyme) prior to shearing and PCR but after ligating barcodes? But what could occur is that one species get a higher number of reads than the other?Comment
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Right. We would normally pool the two species after the shearing and second ligation, but you could do it before.Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.comComment
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Great, thanks again. Our initial data shows reads on all 48 barcodes so I hope we will get some data from most. You've been really helpful!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.Today, 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... -
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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