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Is there an equation, based on the finished library fragment size, for calculating the molar amount of ssDNA that should be added to one lane of a flowcell?
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(1500 x DNA concentration in ng/ul) / library size in bp = nM concentration -
I think he's looking more for X pM = Y c/mm^2.
And I think the answer is there is no good answer.Comment
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Maybe kmpphd would like to weigh in: did you get the answer you were seeking?Comment
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Thank you both for the responses. I should have been more explicit in stating my problem, though GW saw where I was going. I was seeking a way to relate pmoles of library fragments of a certain length to the amount of "real estate" that would eventually be taken up by clusters. For this discussion let's assume a single molecule "cluster," and ignore DNA flexibility. To some approximation, Cluster Area = (# of clusters <in some way related to pmoles of frag>)[3.14(length of frag)^2], right? Based on this rationale, if we then achieve optimal cluster density using X pmoles of a 200 nt fragment, wouldn't we be in the ballpark using 0.25X pmoles of a 400 nt frag?
If equations don't help, as I assume from GW's further comment, what adjustments do you make for different library fragment lengths?Comment
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Okay, I get what you're driving at. I think there are several real-world factors that will skew the theoretical calculation. First, the DNA is flexible, so there will be an effective length that's not linear with actual length. Second, the practical upper limit on cluster density is determined primarily by the ability of the imaging software to resolve individual clusters. Any calculation should take into consideration the software, camera resolution, and detector sensitivity (fluorescence intensity decreases as the area increases). Third, the clusters are distributed randomly instead of evenly, and the probability of cluster overlap is not linear with either cluster number or area.
As a practical matter, we haven't observed much difference between libraries ranging in size from 100bp (some people love to sequence adapter dimers!) to 350bp when loaded at the same molar concentration.Comment
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Cluster density may be influenced by the density of primers anchored to the cell surface. The paper describing chemistry of primer attachment is vague about this and it is possible it this was empirically optimized.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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