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I'm just wondering if an experiment run using say 50bp reads and that requires a sequencing depth of 200 million reads to fully assess gene expression, what affect will using 100bp reads have on that sequencing depth? Obviously less sequencing depth would be required, but I was wondering if anyone could give me a rough estimate of how much?
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When using RNA-Seq to study gene expression read length is not a significant factor; what matters is read counts. Whether you align 100bp paired reads or a 50bp single read to a gene they each still only count as one read. It is counts that give you analytical power in expression studies, not how long your alignments are.Originally posted by bob-loblaw View Post -
What kmcarr is saying is correct; counts mean more than the read length in expression analysis. However, a paired end read 2x50bp will be more beneficial than a 100bp single read as it enables the ability to remove PCR duplicates with more precision.Originally posted by kmcarr View PostComment
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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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