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Old 04-05-2014, 11:31 AM   #1
zrl
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Default When to amplify libraries for RNA-seq?

Hi,

I have a relatively basic question. I am performing RNA-seq on some small tissue samples and I have constructed my first set of libraries for Illumina sequencing using the Ovation/NuGen SPIA system. My libraries are at about 2ng/ul concentration, or around 6nM. My question is: how do you known when to do PCR amplification of your libraries? I think that my RNA is concentrated enough to cluster directly onto a flow cell, but I have been told that amplification is usually necessary for SPIA. On the other hand, I would like to reduce amplification bias as much as possible.

Any recommendations here? Any rough guidelines that you use to determine whether to amplify or not?

Thanks in advance, zrl
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Old 04-05-2014, 12:46 PM   #2
luc
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Sorry, I have no experience with SPIA. If you can confirm your library concentration with qPCR (nanodrop, Qbit, and bioanalyzer are not good enough), PCR-free sequencing should be possible.
What is the purpose of your experiments? In case you want to assess any kind of DGE, you should use protocols that you can maintain unchanged for future samples.
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Old 04-05-2014, 04:46 PM   #3
zrl
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Thanks for your input, luc. I do plan to look at DGE and will make sure to use a consistent protocol between all samples. I have only quantified with Qubit, and plan to use Kapa qPCR to validate.
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Old 04-07-2014, 05:07 PM   #4
zrl
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For the information of anyone else using SPIA: I have spoken to a couple researchers who have had significant difficulties sequencing their libraries when they have not done a PCR amplification following library prep. PCR helps enrich for fragments with both adapters, which might be the explanation.
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Old 04-07-2014, 06:17 PM   #5
jwfoley
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How did you add the sequencing adapters to the library after SPIA? Did you then select for successful products with adapters on both ends? Normally PCR takes care of that for you by diluting out the failures, but if you do no PCR then you have a problem, especially if you used ligation, which is very inefficient. Perhaps your library can still be sequenced, but at the very least you need to quantify sequenceable molecules in your product (by qPCR) because this will be less than the total mass. KAPA has a nice kit with standards for this.
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Old 04-08-2014, 06:59 AM   #6
zrl
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Quote:
Originally Posted by jwfoley View Post
How did you add the sequencing adapters to the library after SPIA? Did you then select for successful products with adapters on both ends? Normally PCR takes care of that for you by diluting out the failures, but if you do no PCR then you have a problem, especially if you used ligation, which is very inefficient. Perhaps your library can still be sequenced, but at the very least you need to quantify sequenceable molecules in your product (by qPCR) because this will be less than the total mass. KAPA has a nice kit with standards for this.
Thanks jwfoley. I did use ligation. I think that I now understand why PCR is necessary for enrichment of successful products, as well as why qPCR is also crucial. I do plan to use the Kapa kit to quantify.
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