Nextera insert sizes larger than expected

[fanli]

I believe we use Qubit or Bioanalyzer for post-library quant and pooling, but I'll ask the bench person to chime in. But more or less, yes, libraries that look outwardly identical in terms of fragment size and concentration do not cluster similarly at all.

[pmiguel]

Originally Posted by fanli

Yeah, we haven't had any issues with the C1 libraries. Our issues actually come from bulk cell (albeit low input) RNA-seq and shotgun metagenomics libraries.

BTW, your cDNA is double-stranded DNA, right?

We had someone give us some "cDNA" and it was, I guess, but it was apparently just 1st strand cDNA. With the DNA strand still annealed to the RNA (template) strand.

We thought we were okay because we used a Qubit and a double-stranded fluor to measure the concentration to put into the reactions.

But, near as I can tell, even Invitrogen's fancy double-stranded DNA fluor doesn't distinguish between dsDNA and a DNA/RNA hybrid... (Does anyone know?)

Also, near as I can tell, Tn5 doesn't see DNA/RNA hybrids as a template for transposition. Or, if it does, Nextera doesn't create anything PCR-able from such a starting point.

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Phillip

[fanli]

Originally Posted by pmiguel

BTW, your cDNA is double-stranded DNA, right?

We had someone give us some "cDNA" and it was, I guess, but it was apparently just 1st strand cDNA. With the DNA strand still annealed to the RNA (template) strand.

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Phillip

Yes, for the low-input RNA-seq we used Clontech Ultra Low Input RNA kit to make dsDNA (protocol here).

[pmiguel]

Originally Posted by fanli

I believe we use Qubit or Bioanalyzer for post-library quant and pooling, but I'll ask the bench person to chime in. But more or less, yes, libraries that look outwardly identical in terms of fragment size and concentration do not cluster similarly at all.

You might have to use qPCR for these, then.

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Phillip

[fanli]

Phillip,

Thanks for the suggestions. We're going to do the Kapa qPCR to see what's happening. I'll keep the thread updated on our findings.

Best,
Fan

[rudzik79]

I know that this post is dead for 3 years now, but I had the same problem and I know that many people are looking for an answer. What they are telling me is that tagmentase is an extremely labile enzyme and it is enough for it to be a little bit outdated or to have several freeze-thaw cycles to lose most of its activity. Hence very large library sizes - tagmentation is just not good enough, and increasing its duration will not help because the enzyme is inactive. Better switch to other methods of DNA shearing.

[pmiguel]

Originally Posted by rudzik79

I know that this post is dead for 3 years now, but I had the same problem and I know that many people are looking for an answer. What they are telling me is that tagmentase is an extremely labile enzyme and it is enough for it to be a little bit outdated or to have several freeze-thaw cycles to lose most of its activity. Hence very large library sizes - tagmentation is just not good enough, and increasing its duration will not help because the enzyme is inactive. Better switch to other methods of DNA shearing.

Eh? Who are the "they" to whom you refer? Nextera doesn't seem particularly labile to me.

One point of possible confusion: after tagmentation, but before PCR, everything tends to look very big because the Tn5 has inserted but apparently it still tethering both DNA ends to each other. See here for more details:
http://www.ncbi.nlm.nih.gov/pubmed/25326703
This paper describes a "contiguity preserving transposition based sequencing" method that takes advantage of this behavior of Tn5.

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Phillip

[GenoMax]

Originally Posted by rudzik79

I know that this post is dead for 3 years now

Really Last posting in this thread was on 20th April of this year by @fanli

[pmiguel]

Originally Posted by GenoMax

Really Last posting in this thread was on 20th April of this year by @fanli

Yes, I think it is possible that rudzik79 did not read the entire thread prior to posting. Which one could understand, I suppose. But in this case led to an erroneous assumption as to the "aliveness" of the thread...

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Phillip