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Old 08-12-2011, 11:54 AM   #1
flyingoyster
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Smile RADseq

Anyone knows well about Restriction-Associated-DNA sequencing? I read a lot of papers about this. It seems that this will produce thousands of markers in very short time. I wonder someone here is very familiar with RADseq or have ever done this before. I may need to do some work on RADseq. If someone can talk about how to design this, that would be great! Thanks,
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Old 08-12-2011, 01:51 PM   #2
jsteed
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I have been researching RAD sequencing myself, take a look at this website
https://www.wiki.ed.ac.uk/display/RADSequencing/Home
I have found it very useful.
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Old 04-18-2012, 08:04 AM   #3
maureen
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You can join the RAD mailing group. The info is on the above wiki.

We use RAD in our lab and it's amazing. Good luck!
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Old 06-12-2012, 04:28 PM   #4
jboone123
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Lots of resources available for RAD throughout the world. The wiki provided above is one place. Also many publications and reviews on the subject. Feel free to contact us at Floragenex if you have any questions as well.
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Old 10-09-2012, 10:03 AM   #5
Gana
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I am having problem with RadSeq library prep. There is no library being amplified. Even though I titrated P1 primer amount down to 1/100X from the suggested amount only multimers of P1 adapter primers are being amplified. Why P1 is not being ligated to DNA although it seems they concatenate with each other well?
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Old 10-10-2012, 06:07 AM   #6
pmiguel
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Quote:
Originally Posted by Gana View Post
I am having problem with RadSeq library prep. There is no library being amplified. Even though I titrated P1 primer amount down to 1/100X from the suggested amount only multimers of P1 adapter primers are being amplified. Why P1 is not being ligated to DNA although it seems they concatenate with each other well?
Are you kidding? If you want some chance of getting a useful answer, you need to provide more detail about both your library construction methodology and the assay you used to determine that construction was failing in some way.
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Old 10-10-2012, 06:34 AM   #7
Gana
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I followed the protocol from "SNP Discovery and Genotyping for Evolutionary
Genetics Using RAD Sequencing" by Paul D. Etter, Susan Bassham, Paul A. Hohenlohe, Eric Johnson, and William A. Cresko. I working with pine and poplar genomic DNA and digesting with SbfI. All Oligo primers were designed and synthesized following the sequence and modification shown at https://www.wiki.ed.ac.uk/display/RADSequencing/Home.

After ligation of P1 (titrated down to 1/100X suggested amount pooled DNA is sheared using Covaris with 400 bp peak. Desired bands of 300-600bp size get purified from agarose gel, end-repaired using the NEB End-repair kit and dA tailed using NEB klenow exo with rATP. After P2 is ligation library get amplified using Phusion HF. Amplified library run on the agarose gel and there are only multiple bands of distinct sizes (supposedly multimers of P1 adapter). We purified bands around 400 bp and sequenced it on Mi-Seq and all we got was adapter sequence. So I do not know why it is not working. I know that my poplar DNA is good quality and they worked fine for SureSelect library construction.
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File Type: jpg 2012-10-9 Poplar Rad-Seq.jpg (70.6 KB, 40 views)
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Old 10-10-2012, 07:36 AM   #8
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What are the sequences of your two P1 adapter oligos?

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Old 10-10-2012, 07:45 AM   #9
Gana
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The first 12 primers from the "P1 adapters" list at https://www.wiki.ed.ac.uk/display/RADSequencing/Home.
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Old 10-10-2012, 08:25 AM   #10
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What about the other strand of the P1 adapter?

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Old 10-10-2012, 08:51 AM   #11
Gana
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From the same source.
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Old 10-10-2012, 08:58 AM   #12
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Did you do QC at any other step. Like an Agilent high sensitivity chip prior to PCR?

Generally when adapter dimers dominate a library it is because the effective ends-insert ratio is too high. So if you lost most of the sheared DNA at some step, or the end repair failed, that might explain what is going on.

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Old 10-10-2012, 09:16 AM   #13
Gana
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There is no QC step until I run check gel after PCR. I do take concentration on NanoDrop after each purification step (Qiagen) to make sure I have DNA. I had DNA clearly after shearing and before End-repair.

I didn't think that End-repair might have failed since it was a kit purchased from NEB but I will definitely check on this step.
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Old 10-10-2012, 10:00 AM   #14
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Nanodrop is typically a terrible way to quantitate genomic DNA. Fairly normal for DNA preps to read 10x their actual concentration because of RNA and degraded RNA.

Also easy to lose your DNA during an extraction from an agarose gel.

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Old 10-10-2012, 10:19 AM   #15
Gana
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We are aware of these problems and I am quite confident that agarose gel extraction gives me at least 50-75% yield. The same DNA is working for other library construction so quantification is not a big issue. But I am testing your suggestion and using a different End-repair kit and will do the dA tailing, P2 ligation, and PCR again. Besides gel extraction and quantification problem if there is anything I might be missing? I do appreciate your comments. I am new to NGS and library construction and still learning.
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Old 10-11-2012, 06:57 AM   #16
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Well, if you are getting a high number of "adapter dimers" -- P1-P2 ligation products with no, or very short insert it becomes an issue. Basically they are the devil to get rid of after enrichment PCR. First they will amplify preferentially during PCR, because they are small. Second, even if you do a size selection after PCR, you can't remove all of them, because some of them will anneal back to full length library molecules.

That is why I would recommend adding a QC step after P2 ligation. If you see some P1-P2 (adapter dimer) length products at that point, I suggest an additional size selection. Ampure would probably do the trick.

In principle, you could do the size selection after enrichment PCR. But you would want to force the "hitch-hiking" adapter dimers to leave their "hiding place" by running a denaturing gel. But I have never tried this -- probably more trouble that it is worth. Multiple cycles of ampure also help. But this would be for cases where less than 50% of the amplicons are adapter dimers.

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Old 10-11-2012, 07:35 AM   #17
Gana
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What do you think is the cause of these unwanted passengers? Titration of P1 down is not helping. So there must be something that is not optimal for either P1 or P2 ligations. Could you think of anything ...? Will titration of P2 make any sense?

Before optimizing any of these two ligations there is no point adding a QC step or using expensive AMPure beads. If I get a library that is contaminated then AMPure might be a choice but if there is no library amplification then .
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Old 06-13-2013, 07:30 PM   #18
suzhebeibei
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Default please help me!

does anyone here do the detection of GMO using RADSeq?
am very hurry to do this project but have no idea about it.
how to use RADSeq to detect GMOs?
i will be appreciated if anyone can talk with me about this!
thx!
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Old 06-13-2013, 07:48 PM   #19
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suzhebeibei, RAD-Seq produces sequences from random loci across a genome. It is not usually used to check for the presence of a single region. Would a PCR test work instead?
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Old 06-13-2013, 08:44 PM   #20
suzhebeibei
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Quote:
Originally Posted by SNPsaurus View Post
suzhebeibei, RAD-Seq produces sequences from random loci across a genome. It is not usually used to check for the presence of a single region. Would a PCR test work instead?
i know,but we can choose the enzymes to control this situation.
The most urgent things now is that i don not konw how to analyse the data sequenced.would u help me about that?
PCR is very useful,but it is also time-consuming,low through-put,so we want to search for a innovation method to solve the problem.
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