[chariko]

A library with average size up to 2000 is no problem. See our just published paper in ACS Synthetic Biology for some tips. The length of time of tagmentation is not important as long as it goes to completion, i.e. all the transposomes have tagmented the DNA sample. Tagmentation is stoichiometric, not catalytic The amount of DNA going into the tagmentation reaction is critical. Too much DNA will give fragments too large to be amplified and too little DNA will give small fragments that will be lost during SPRI. If you are using the Nextera XT kit and protocol, the extension time should be fine. Most likely you need to quantify and dilute your DNA more carefully.

Thanks for your answer I will take a look at your paper (reference?).

Anyway, DNA was quantified with Qubit prior to being diluted so I suppose everything was correct but I will check it again...

Does anyone have any other clues?

[nucacidhunter]

There could be many reasons, but considering Bioanalyzer trace I would check following:
1- issues with pipetting most likely calibration which would cause errors in quantification and normalisation

2- adequate mixing of reactions

[anamar]

Actually, I think that sample 2 has some >12 kb stuff in it that ended up running into sample 3. Which may sound crazy, but over time I have come to the conclusion that some lanes share part of the same paths. So if they do not completely clear, high molecular weight stuff from an earlier well can end up in a later one.



--
Phillip

Hello Phillip, Doing my library prep and after amplification with SMARTER kit I am almost certain that some huge molecules move to the next well in the bioanalyzer HS chips. I know this is a very old post from you but have you tested this and/ or have alternative explanations, Best, Ana

[M4TTN]

Hi anamar,

I am pretty sure that all the Bioanalyser lanes converge into one area on the chip - at the point that they are "analysed". Each sample reaches there at different times based on the relative lengths of the paths on the Bioanalyser chip.

So, yes, if what I just stated is correct, I think it is entirely possible that very large molecules could migrate so slowly that they "contaminate" the lane trace of the next sample that passes through the analysis/quantification area.

[fanli]

A library with average size up to 2000 is no problem. See our just published paper in ACS Synthetic Biology for some tips. The length of time of tagmentation is not important as long as it goes to completion, i.e. all the transposomes have tagmented the DNA sample. Tagmentation is stoichiometric, not catalytic The amount of DNA going into the tagmentation reaction is critical. Too much DNA will give fragments too large to be amplified and too little DNA will give small fragments that will be lost during SPRI. If you are using the Nextera XT kit and protocol, the extension time should be fine. Most likely you need to quantify and dilute your DNA more carefully.

@creeves, can you elaborate on why the tagmentation is stoichiometric? If I understand correctly, it is cut and paste - so you eventually run out of adapter sequence to "cut"? Quoted from http://genome.cshlp.org/content/24/12/2033.full:

Transposition works through a “cut-and-paste” mechanism, where the Tn5 excises itself from the donor DNA and inserts into a target sequence, creating a 9-bp duplication of the target

[luc]

Fanli,
the transposase in the kit does not have any donor DNA available to it. The enzyme needs to "loaded" beforehand with oligos; thus each enzyme molecule can cut only once (? or twice?).
The appeal of the Nextera protocol for most applications is an enigma to me.

[fanli]

@luc, Thanks that makes more sense. Seems to be a really inefficient use of enzyme though IMO. For future reference, here's another helpful link:

http://bitesizebio.com/13567/too-good-to-be-true-what-can-nextera-do-for-you/

We got sucked into Nextera from the Fluidigm -> Clontech protocol. :/

[pmiguel]

@luc, Thanks that makes more sense. Seems to be a really inefficient use of enzyme though IMO. For future reference, here's another helpful link:

http://bitesizebio.com/13567/too-good-to-be-true-what-can-nextera-do-for-you/

We got sucked into Nextera from the Fluidigm -> Clontech protocol. :/

What problem are you having? The Fluidigm FC1 protocol generates cDNA that seems to work well with the 1/4 Nextera reactions recommended by Fluidigm.

Did you determine the amount of cDNA to add using fluorimetry?

For 1-2 96-well plates the Nextera protocol is fast and effective. Of course if you are doing one of those new 600 well Fluidigm chips it would be a substantial amount of scale-up...

--
Phillip

[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.

Not to get too off-topic, but we end up with fragment sizes that are somewhat longer than optimal for Nextseq 500, but similar size to the C1 libraries. Loading the recommended concentration (~1.8pM) gets us good cluster density, etc. for C1 libraries, but grossly underclustered flow cells for the other two types (19K/mm2 for our last run of RNA-seq). Long story short, we're trying to figure out why this is happening and just got thinking about the tagmentation. But if the fragment distribution after tagmentation is similar...then they should cluster similarly on a flow cell right?

[pmiguel]

So qPCR (eg, KAPA) titration isn't predicting the cluster density correctly?

--
Phillip

[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]

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.

--
Phillip

[fanli]

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.

--
Phillip

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

[pmiguel]

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.

--
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