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Old 10-08-2014, 10:36 AM   #1
liumangmang
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Default PCR step in DNA library prep?

Hi,
I have a question about PCR step in library prep. We are using NEBNext kit and we want to pool several samples using index.
After size selection, I have about 250 ng DNA (500 bp including adaptor). It's enough in amount but we need to add index so we still need to do PCR.
The problem is if I only get 4 folds of DNA after PCR, does that means 25% DNA do not get indexed?
Will it affect sequencing efficiency or depth?
Could anyone give me some clue? Thanks a lot.
This is the kit I'm using:
https://www.neb.com/products/e7370-n...t-for-illumina
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Old 10-08-2014, 08:13 PM   #2
weigrc
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It is normal to have less than 40% of input DNA recovered as adapter-ligated molecules; for those DNA who don't have a pair of adapters, can not be sequenced. Using QPCR for calculating the library molecular before sequencing.

And you may also use QPCR to estimate how much library molecules in the 250 ng and after PCR, then you can see if it is reasonable for the PCR amplification efficiency.
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Old 10-09-2014, 02:16 AM   #3
nucacidhunter
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Quote:
The problem is if I only get 4 folds of DNA after PCR, does that means 25% DNA do not get indexed?
Will it affect sequencing efficiency or depth?
How many PCR cycle was done and I wonder on what bases you think that 25% of amplicons are getting indexed. During PCR one would expect doubling the amount each cycle if efficiency is 100% and all template is amplifiable (have primer binding sites). IF you have done 10 cycles and only see 4 fold increase in DNA amount it could be either problem with PCR or that the ligation was poor so all fragments were not amplifiable. I would be interested to know how the quantification was done before and after PCR.
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Old 10-09-2014, 08:00 AM   #4
liumangmang
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Hi,
In order to figure out what's wrong, I did two tubes in parallel:
One with 25ng template VS The other with 250 ng template
Template is the DNA recovered after adaptor ligation and AMP bead size selection (~500bp including adapotor). Other reagents are the same between two tubes (as written in the kit protocal).

I did 9 cycles of PCR. Then I cleaned up with 1X AMP beads to get ride of small pieces (such as primers.etc).

I measured dsDNA concentration after clean up. The tube has 25ng template gave me about 32 folds of DNA and the tube has 250ng only gave me about 1.8 fold. So the PCR with more template worked poorly.

Although in theory 5 cycles should be enough to amplify 32 folds, but as weigrc said, not all DNA fragment I put in has a pair of adaptor ligated.

I asked people of NEB tech support and also people of sequencing center. They kinda gave me different answers.

This is what I got back from NEB:
"I think the issue with your PCR reactions is over-amplification, which generally means over-cycling of a PCR reaction after primers are all used up. There are several characterics associated with over-amplification:
1. Larger than expected fragment sizes: When primers become limiting during the PCR reaction, fragments with different inserts anneal through the adapter sequences at the ends. This results in DNA molecules with a bubble shape or daisy chains of several DNA molecules. These bands typically start at two times the expected size for your library.
2. library bias: Please refer to the attched reference for more details.
3. Decreased library yields: This is likely due to the strong 3'-5' exo activity of Q5, which starts to degrade DNA when primers run out in the reactions.

The best way to avoid over-amplification is to minimize the number of PCR cycles. Although it might look like you have 200-300ng DNA template after ligation and size selection, not all DNA fragments are amplificable. The DNA pool actually consists of 3 groups: original input DNA in this size range, DNA fragments with one adaptor ligated to one end, and DNA fragments with two adaptors ligated to both ends. Only fragments with adaptors on both ends can be amplified with index primers, and in theory, 2 cycles are enough to add index and Illumina sequences to the libraries. In your case with 1µg input, I would recommend < 4 cycles to avoid over-amplification and its associated bias. "

I think his point is that in order to add index, I only need to amplify 4 folds of DNA and reduce number of cycles would help enhance PCR efficiency???

In contrast, the people in sequencing center suggest me to have at least 32 fold after PCR, so that the untagged DNA will be less than 3%. And they also recommend to use ~20ng template (which is consistant with my test run).

So I'm still thinking whose opinion I should listen to......I tend to believe the guy from sequencing center because I think if a big portion of DNA do not have index, they just occupy and waste the sequencing reads and there's no way I could tell which sample it comes from when 12 samples are mixed together in one lane.

I appreciate anyone could share more opinions.
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Old 10-09-2014, 08:09 AM   #5
liumangmang
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BTW, I used Qubit Fluorometer to measure dsDNA concentration before and after PCR. And I have ran Egel to check the size of PCR products.
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Old 10-09-2014, 03:16 PM   #6
nucacidhunter
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Both Techsupport and sequencing center comments are correct. However, each will give different end results. If higher amount of DNA is used for lower number of cycling, diversity of library will be higher (low duplication) and coverage will be more uniform.
NEB kit ligates only partial Illumina adapters to fragments which lacks flow cell binding motives. Those motives and index are added during PCR. Therefore, fragments that are not amplified by PCR will not bind to flow cell and will be washed away during cluster generation. If your sequencing centre uses qPCR for quantifying libraries prior to clustering there will be no harm from non-amplifiable fragments because primers complementary to flow cell binding motives are used for amplification.

Do the size distribution of libraries on gel looks different?
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