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Hello, I have a basic question. I am extracting samples that will be around 3.5 Mb and only done sonication on whole genome. Can I still use it with the smaller input or will I over fragment? Is enzymatic a better way to go?
Thanks
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Are you using Covaris? I think it wouldn't be a problem because you can regulate many things with the settings of the machine. I myself sonicated fragments around 100 to 10 kb end ended in the library range (300bp) with the settings given by Covaris. -
Large DNAs will fragment during isolation, so after isolation your 3.5 Mb DNA will most likely have the same fragment size distribution as you would get from purifying larger genomes. There should be no difference in further fragmentation (Covaris, other sonication, etc.) between the two. So, short answer, you shouldn't need to change any conditions for fragmenting.
Where things get tricky is on the low end. In our hands, Covaris works pretty well with fragments as small as ~1 kb, but smaller than that and you start to run into problems. 400 bp amplicons really don't fragment at all in the Covaris. I'm sure there is a good biophysical explanation for this, but we have just seen this empirically.Comment
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Yes, planning on using Covaris. Thanks so much for your help!Comment
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You may also want to try Fragmentase from NEBComment
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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...06-18-2026, 07:11 AM -
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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