[cement_head]

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?

[eperry]

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?

[Tom Quinn]

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

[cbird]

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.

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 .

[SNPsaurus]

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.

[rosatoc]

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!

[suzhebeibei]

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!

[suzhebeibei]

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.......

[SNPsaurus]

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.

[suzhebeibei]

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.

[SNPsaurus]

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?

[suzhebeibei]

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!

[Gana]

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 .

[pmiguel]

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.

--
Phillip

[Gana]

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.