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Old 06-14-2013, 12:52 PM   #21
SNPsaurus
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If you pick an enzyme that digests the DNA of the GMO material, then the RAD-Seq library will have that sequence plus all the other genomic loci that have the cut site. So to analyze the data you could take the RAD-Seq reads and align them to a reference sequence of the GMO. I am still unsure if it offers any advantage over existing methods, though.
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Old 06-14-2013, 10:31 PM   #22
suzhebeibei
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Originally Posted by SNPsaurus View Post
If you pick an enzyme that digests the DNA of the GMO material, then the RAD-Seq library will have that sequence plus all the other genomic loci that have the cut site. So to analyze the data you could take the RAD-Seq reads and align them to a reference sequence of the GMO. I am still unsure if it offers any advantage over existing methods, though.

yeah,you are right.and this is where i also feel confused.
when i get the sequence,i will firstly align them to the insertion sequence,and pick up the correct reads,and then align them to the genome to pick up the other correct reads.
all of these reads are what we want to analyse the insertion sites,but the question is how should i analyse these datas,and which kinds of softwares i should use,confused........i have never known about bioinformation before,so.......
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Old 06-15-2013, 05:58 AM   #23
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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.

hello,i have searched the RADSeq home page,and i want to know how to use the Stacks,can u help me?thx a lot!
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Old 07-24-2013, 11:26 AM   #24
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Gana, I am confused why you shear (Corvaris) your sample. For genotyping by sequencing (Elshire protocol) the restricted fragments are ligated to the barcoded adapters and then amplified with Illumina primers. A titration experiment is run (for each enzyme/species combination) to determine the optimal adapter concentration to use for library construction. If this is old news I apologize, but I would like to know the value of shearing. Thanks!
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Old 07-24-2013, 11:37 AM   #25
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rosatoc, I thought Gana was carrying out RAD-Seq, not GBS. RAD-Seq shears the genomic DNA after ligation of the first adapters so that there are a full set of markers at every fragment size, avoiding any problems from having different fragment size ranges selected between library preps of different sets of individuals.
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Old 10-18-2013, 12:15 AM   #26
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Gana, your problem probably started with the use of the NanoDrop right at the beginning when you were determining how much DNA to digest. Use a fluorescent plate reader to quant your DNA.

Also, we've put a biotin on the end of the p1 adapters, which when used in combination with streptavidin-coated dynabeads, allows us to visualize ligation efficiency using simple gel electrophoresis.


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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 09-29-2014, 01:56 PM   #27
Tom Quinn
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Originally Posted by suzhebeibei View Post
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!
You can do this for many GMOs using this kit from BioRad. It uses PCR to amplify sequences frequently inserted as GMOs are made.

http://www.bio-rad.com/en-us/product...vestigator-kit
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Old 02-10-2015, 02:49 PM   #28
eperry
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Default RADseq new cut sites?

Does anyone have a script that can use aligned reads from RADseq libraries to identify putative mutations that have created new cut sites or disrupted cut sites between samples?
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Old 05-24-2018, 01:14 PM   #29
cement_head
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For RAD-Seq libraries, to be sequenced on Illumina MiSeq, what concentration do people typically use? Something akin to 16S metagenomics sequencing? I.E. 4.0 pM with a 10% spike-in of PhiX control?
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