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Does anyone have any experience with the RPKM (reads per kilobase of exon per million mapped reads) as calculated by TopHat? I can't find any documentation on how this is calculated, and I'm trying to make sure that it standardizes for the total number of mapped reads (as suggested by the name of the metric). I would like to compare RPKM values between samples which have different numbers of total reads and mapped reads, and I'm wondering if I need to do my own standardization for the total number of mapped reads or if this is unnecessary because TopHat has already corrected for it.
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Please note that the RPKM calculation was an experimental feature that is now considered deprecated with the release of Cufflinks. I strongly recommend you discontinue using TopHat to calculate it, and move to Cufflinks instead. The RPKM code will be removed from TopHat in the next release.
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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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