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I am going to be tracking changes in methylation status of cells over time and with varying treatments. For MBD seq do I need to sequence a corresponding input reaction for every time point and treatment or can I compare the different groups to each other instead? I will be analyzing untreated time point one as a baseline. Would this suffice?
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In general when we're doing this type of enrichment work we only do a single input sample using the same conditions as for the enriched samples. Most of the artefacts come from the DNA preparation, mapping and sequencing stages and won't change between timepoints or treatments (unless you have a treatment which is going to grossly change the structure of your DNA in some way). Doing a matched input for each sample would get really expensive really quickly!
In some cases we don't bother doing an input at all but just use data from previous inputs we've run to either use in the analysis or to provide sets of regions which we should ignore.
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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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