Hi all,
Our lab just did our first Nextera XT run on a MiSeq. I'm just the informatics guy and didn't actually prep the libraries myself, but the person who did says the protocol is nicely straightforward compared to some earlier library protocols.
The resulting run, unfortunately, had a fairly wide range in sample abundances and I'm wondering if any of you have insight into why or what we might do differently to achieve uniform abundances? I've pasted a histogram of per-sample read counts below. Input material was normalized to 1ng/sample as measured with flourimetry (picogreen or qubit).
There is a 10-fold difference between the least abundant sample and the most abundant. My (very limited) understanding of the protocol is that normalization happens at the last stage of library prep where tagmented samples are loaded with magnetic beads and shaken at 1800rpm. A limited quantity of sample material is supposed to saturate the binding capacity of the beads in each tube. When the material is released from the beads one obtains about the same amount of material from each sample.
Does that sound right? Any ideas on what we could do differently to achieve better normalization? Are the results I have here considered "good" normalization?
Our lab just did our first Nextera XT run on a MiSeq. I'm just the informatics guy and didn't actually prep the libraries myself, but the person who did says the protocol is nicely straightforward compared to some earlier library protocols.
The resulting run, unfortunately, had a fairly wide range in sample abundances and I'm wondering if any of you have insight into why or what we might do differently to achieve uniform abundances? I've pasted a histogram of per-sample read counts below. Input material was normalized to 1ng/sample as measured with flourimetry (picogreen or qubit).
There is a 10-fold difference between the least abundant sample and the most abundant. My (very limited) understanding of the protocol is that normalization happens at the last stage of library prep where tagmented samples are loaded with magnetic beads and shaken at 1800rpm. A limited quantity of sample material is supposed to saturate the binding capacity of the beads in each tube. When the material is released from the beads one obtains about the same amount of material from each sample.
Does that sound right? Any ideas on what we could do differently to achieve better normalization? Are the results I have here considered "good" normalization?
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