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Old 06-28-2011, 07:33 AM   #21
pmiguel
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Originally Posted by ETHANol View Post
The slower running fragments after amplification is from 'hybrid' fragments, i.e. fragments that are created after primer becomes limiting and the DNA fragments anneal by the adaptors sequences at the ends but not in the 'unique' sequences in the middle. Think of a bubble structure.
[...]
Does not fit all the data I have seen. I have seen cases where higher order multimers are formed. That is, a 1-mer peak (expected length), 2-mer peak (a little less than double the expected length), 3-mer, etc.

This would fit with a daisy chain hypothesis but not with a bubble hypothesis.

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Old 06-28-2011, 07:52 AM   #22
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My guess is that all 4 of these are at work in various situations. The length of the TruSeq adapters is probably the source of these issues. What about mitigation?
Phillip, great post and nice explanations. It popped up while I was writing mine (I'm doing an experiment so it took a while).

As with everything in biology, you have to be correct in that it's all four at work depending on the situation.

I'm pretty sure that what I see are 'bubbles' because: 1) if I take an amplified library and denature it and let it reanneal the products are found at the same larger size, 2) if I submit the library to one more cycle of PCR with fresh primers the fragments return to the correct size, and 3) If I reduce the number of cycles I don't get the larger products. In my case it all adds up to being 'bubbles'

The strange thing that kind of threw me off at first is that the larger products are exactly twice the size of the 'correct' sized products.

I started getting the larger products when I switch to the Kappa polymerase. Turns out you have to to a lot less cycles then with Phusion polymerase.
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Old 06-28-2011, 08:02 AM   #23
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Does not fit all the data I have seen. I have seen cases where higher order multimers are formed. That is, a 1-mer peak (expected length), 2-mer peak (a little less than double the expected length), 3-mer, etc.

This would fit with a daisy chain hypothesis but not with a bubble hypothesis.

--
Phillip
Actually, now that I think about it more, the Daisy Chain and Bubble hypotheses could both support multimers beyond dimers. Probably the best way to distinguish between them would be to take half a sample you know is showing the phenomenon and treat that half with S1 nuclease. The run the treated and untreated on the bioanalyzer.

The Daisy Chain hypothesis posits double-stranded amplicons linked end-to end by adapter-ends annealing. So either S1 would do nothing, or slice off the inter-amplicon annealed ends. Resulting in slightly shorter than 1-mer peak.

The Bubble hypothesis would leave much of the multimeric amplicons single-stranded and allow them to be nearly completely degraded by S1. So the 1-mer peak would be the same, while the multimer peaks would be destroyed.

The "PCR Overamplification" hypothesis would result in no change after S1 nuclease digestion because the multimers would be completely double stranded.

My guess is that a mixture of all possible multimer formation modes are happening.

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Old 06-28-2011, 08:18 AM   #24
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Phillip, great post and nice explanations. It popped up while I was writing mine (I'm doing an experiment so it took a while).
Hilarious. Looks like we are writing posts at the same time and converging to many of the same conclusions.

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As with everything in biology, you have to be correct in that it's all four at work depending on the situation.

I'm pretty sure that what I see are 'bubbles' because: 1) if I take an amplified library and denature it and let it reanneal the products are found at the same larger size,
Interesting! We did one where we were seeing multimers and a high molecular weight smear for a 454 amplicon library. Heated the samples to denaturation temp, then turned off the thermal cycler. They slowly cooled to ambient. The multimers disappeared.

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2) if I submit the library to one more cycle of PCR with fresh primers the fragments return to the correct size, and
Just fresh primers, or a whole new set of PCR reactants? Thing is, this doesn't really distinguish among the hypotheses. Daisy chaining probably becomes favorable under the same conditions that Bubble Product formation would -- high concentration of products with respect to primer concentrations.

And, your fix, another cycle, would work for the PCR overamplification hypothesis as well. (PCR favors shorter templates.)

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3) If I reduce the number of cycles I don't get the larger products. In my case it all adds up to being 'bubbles'

The strange thing that kind of threw me off at first is that the larger products are exactly twice the size of the 'correct' sized products.
Well, that seems like an argument for Daisy Chaining. I guess a Bubble product might run exactly 2x the size of a normal double stranded product, but I can't think of any reason it would.

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I started getting the larger products when I switch to the Kappa polymerase. Turns out you have to to a lot less cycles then with Phusion polymerase.
We just use the TruSeq reagents. Not sure which polymerase it uses.

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Old 06-28-2011, 10:11 AM   #25
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When I say one more round of PCR what I mean is I take the Qiagen minElute/Ampure purified library and add all the reagent PCR (primers and 2X Mastermix) heat at 98˚ C for 1 min, 60˚ C for 30 sec and 72 ˚C for 1.5 min.

One more thing, the melting temp of the slower then expected running DNA fragments is really low and forms a sharp peak which says that they are only being held together by adaptor interaction.

Daisy chains, bubbles, etc. it's all a little more complicated then I originally thought. The important thing is that the individual DNA molecules are not actually big (at least in my case) and will not interfere with sequencing or quantification by qPCR.
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Old 06-28-2011, 11:04 AM   #26
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[...]

One more thing, the melting temp of the slower then expected running DNA fragments is really low and forms a sharp peak which says that they are only being held together by adaptor interaction.

[...]
Just wondering: what is your assay for melting temp? (I remember the old hyperchromicity plots from long ago. But have not seen one done for decades...)

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Old 06-28-2011, 11:08 AM   #27
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SYBR green on the qPCR machine.

Which reminds me, Kapa makes formulation of their Library amplification polymerase with SYBR green in it so you can monitor library amplification in real time and stop before you over-amplify. I know, I sound like a Kapa sales rep but it solves all these over amplification problems.
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Old 08-01-2011, 08:07 AM   #28
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Hi Phillip,
I'm curious to know if you tried this slow ramp step and if it worked.

-Jen

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How about for the last cycle doing a very slow ramp from 98 oC to 60 oC? That way the full length products will have lots of time to find each other.
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Old 08-01-2011, 08:44 AM   #29
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Thought I'd throw in my experiences with HMW smears. I was preparing libraries using the NEBnext kit and custom barcode PE adapters. The libraries had a normal peak at 350, then a consistent smear going from 500-10kb on bioanalyzer. I usually use small amts of primer (equiv to 0.5 ul 10 nm per 50 ul reaction), so after reading this thread I re-PCR'd with 4 ul of 10 nm primer which completely got rid of the HMW smear. So, +1 for limited primer concentration.
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Old 08-01-2011, 08:59 AM   #30
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edawad, I also had that experience (re-PCR 2 cycles got rid of my HMW peaks). Did you feel that the ultimate easy solution was just to use more primer with every PCR reaction? One time I tried doubling my primer and didn't get any different result, but double may just not be enough. I'm hoping that I can instead add some sort of ramp step or something to my prep (like Phillip suggested) just to get the average size correct moving forward.
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Old 08-01-2011, 09:48 AM   #31
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I did several PCR reactions to test (had enough pre-pcr sample to spare). I tried 0.5, 1, 2, and 4 ul of my 10 nm working soln primer with both 15 and 18 cycles, and only using 4 ul got rid of the smears (which would be 8x more than I usually use for ChIP). Perhaps I had more sample than usual, or my primer soln was too dilute.

Can't answer your question about the ramp step but sounds like something worth trying.
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Old 08-02-2011, 06:03 AM   #32
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Hi Phillip,
I'm curious to know if you tried this slow ramp step and if it worked.

-Jen
Hi Jen,
I never tried it. If I did, I think I would try the same one used in ETHANol's protocol for annealing Y-adapters:
(from:
http://seqanswers.com/forums/showthread.php?t=13093
)


Anneal oligos an a thermal cycler with the following program:
a. 2 min at 95 oC
b. 140 steps of
30 sec at 95 oC (decrease temp 0.5. C every step)
c. Hold at 4. C

If that does not work, I do not know what would.

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Old 08-02-2011, 06:09 AM   #33
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Hi Phillip, I tried this yesterday but did not do such a slow ramp (I did 0.1C/ sec from 98-4C) and got a very strange result..... some of my "500 bp" library split into two humps, one right at 300 bp where it belongs and the other way, way higher. I guess this does suggest that there is some bubble and/or chimera foming. I decided to do another ramp where I simply put the sample on a heat block at 98 and took it off the heater and let it go down slowly overnight at RT. Maybe I'll try this setting as well. Can't hurt. I'm also doing some PCR using 10X the amount of PCR primer. I'll keep you all posted.
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Old 08-02-2011, 09:13 AM   #34
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Hi Jen,
Similarly, when we denatured the sample and then allowed to reanneal at room temp we saw more of the sample end up in the slow peak (whatever that is), not less.

I will be interested to see what your 10X PCR primer result will be.

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Old 12-23-2011, 12:41 PM   #35
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We tried this suggestion last week (slow ramp speed on the last cycle). Our bubble product was also greatly increased, but our concentrations determined by KAPA qPCR were the same as our previous preps that used the TruSeq protocol as written.

Next time I'd like to try increasing the amount of primer. Has anyone done this yet, and what were the results?

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How about for the last cycle doing a very slow ramp from 98 oC to 60 oC? That way the full length products will have lots of time to find each other.
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Old 12-23-2011, 01:48 PM   #36
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In our lab, we've either re-PCRd with 4x primer, or take the bubble'd product and perform an additional 2 cycles of PCR with extra primer. Both of these methods worked to remove the extra HMW product.
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Old 12-25-2011, 01:21 PM   #37
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You can also just ignore the larger peak and go with your qPCR titration.

We usually go with fewer cycles of enrichment PCR now and rarely see the multimer peaks caused by ectopic annealing.

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Old 10-19-2016, 06:40 AM   #38
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Default Using ssDNA and Agilen RNA Chip to solve longer than expected library sizes

Find below an example of what we thought was a library overamplification problem. When the same libraries are denatured at 97 C for 2 minutes and run with RNA chip, the larger fragments disappear. So this supports the bubble and daisy-chain explanation. My conclusion is that if we use qPCR to quantify, these shouls be OK for sequencing and not form e.g. chimeric sequences. Also, reducing number of PCR cycles or adding primers might be a solution. We tried size based selection with AMPure, but it did not work out, which makes sense if this is caused by ssDNA annealing e.g. as heteroduplexes/bubble products.

ssDNABioanalyzer.jpg
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Old 10-19-2016, 07:37 AM   #39
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Find below an example of what we thought was a library overamplification problem. When the same libraries are denatured at 97 C for 2 minutes and run with RNA chip, the larger fragments disappear. So this supports the bubble and daisy-chain explanation. My conclusion is that if we use qPCR to quantify, these shouls be OK for sequencing and not form e.g. chimeric sequences. Also, reducing number of PCR cycles or adding primers might be a solution. We tried size based selection with AMPure, but it did not work out, which makes sense if this is caused by ssDNA annealing e.g. as heteroduplexes/bubble products.

Attachment 4555
Yes, exactly.

Although I've always wondered what a sample would look like after treating it with a single-strand-specific nuclease. That could confirm that there was single-stranded + double stranded regions in the amplicons.

But I've been too lazy to actually do the experiment...
--
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Old 02-21-2017, 12:00 PM   #40
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Hello folks,

Interesting thread! I got a lot of high molecular weight smear in my last run as well (following EMP protocols for soil samples).

I'm trying to figure out the details of the daisy-chain hypothesis:

From what I see the Illumina adapters on either end aren't complementary. So I guess the thought is that a few annealing bps are enough to lead to 3' extension. However, why would this lead to preferential extension to form multimers, thus starting at the adapter sequences? If it is just a few bps duplexing, surely this could happen anywhere within the amplicon.

Is my thinking off. Am I missing a point?
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