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what are thoughts on this new method? no need for a covaris, and low input. interested in using before capture protocol, i am concerned the transposome complex will not yield fragments representative of entire genome.
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what are thoughts on this new method? no need for a covaris, and low input. interested in using before capture protocol, i am concerned the transposome complex will not yield fragments representative of entire genome. -
Moving this to the library prep forum as it's applicable to 454 (and probably solid) as well. -
size distribution using transposition
I heard about the new technology as well. I was just wondering what the size distribution looks like afterwards. As far as I know there is no need to do it. Thanks.Comment
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here is the distribution with bioanalyzer for a full reaction with 10 pcr cycles:
and for 1/5 scaled down reaction with 15 pcr cycles:
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here is the distribution with bioanalyzer for a full reaction with 10 pcr cycles:
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and for 1/5 scaled down reaction with 15 pcr cycles:
http://www.postimage.org/image.php?v=PqE_Lh9[IMG]
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You should check with your capture reagent vendor -- one of the problems seen with Illumina libraries is "daisy-chaining" of fragments via the adapters which brings in off-target material. Nextera uses custom adapters, so any blocking oligos supplied in the kit won't be correct.Comment
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@upenn_ngs: Were this size distributions for Illumina libraries?Comment
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what about ends fragments ?
Hi all,
This fragmentation seems very interesting by its low input DNA and its relative simplicity!
I am wondering about the non representativity of ends fragments with this fragmentation except with a previous step like circularization or adding primers.
Is that makes sense ?
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yes, this is illumina library prep. although there is a great time saving, a fragmentation and ligation protocol with NEB enzyme is still much more cost effective.Comment
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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.
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