Science method

Genotyping - Science method

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Hi all,
I generated mutant lines using the CRISPR/Cas9 method, and my plants are in the T1 generation.
I am trying to send PCR products for Sanger sequencing, but the problem is, the positive control that shows me two bands. I borrowed wild-type genomic DNA from another colleague and got the same result.
I used my primers for genotyping in the previous generation without any problems, so my primers are specific.
I attached my results: Pic 1 shows extracted DNA from leaves by the CTAB method. Pic 2 shows the PCR result of WT and one of the mutant lines. As you can see, I have two bands in the WT lane but only one band in the mutant lane, and Pic3 displays PCR results of other mutant lines with one/two bands. Besides, I did not get any bands for some lines. To check those lines, I used cas9 primers which were successful.
The NC lane is clear, so I don’t have contamination.
I did gradient PCR, changed enzyme, and changed denaturing, annealing and extending time, but they did not work.
I am running out of ideas. What could cause this issue and any suggestions to address it?
Thank you so much in advance for your help!
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Looks like you have non-specific bands. And you are really over-loading your gel - try loading less PCR sample in the wells.
I second the vote to design some new primers. Primers are cheap, endless trouble-shooting is expensive.
Good luck!
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Hello!
I have black C57 DsRed & EGFP mice (from JAX) and need to quantify the expression of EGFP or DsRed at mRNA levels with qPCR. I am wondering if anyone knows about the primers?
Thanks !
P.S
I have the primers for the genotyping of these mice but I think those primers are too big for this purpouse.
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Hi Sina
designing primers for qPCR follow the same rules as for classic PCR, at the exception that amplicon must have 100-150bp length and one of the primer need to be across splice, at most 3' end of the gene..
take a look at the UCSC database, there is a genome browser and typically you can choose to make available the track presenting primers that have been designed and published on the gene (do not forget the rules for qPCR primers). after designing or choosing, you can also test them by in silico PCR on the same site.
all the best
fred
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Hi, I have problems to get a SNP input file working well in Genalex. I tried to change letters by numbers (A=1; C=2; G=3 and T=4) in one column but it does not work. It does not seem to work either using numbers and two columns (codominant). Some help on that ?? In attached, an exemple of my SNP data set. Thanks
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Severine Roques did you ever get SPAGEDI to work on SNPS? I can't seem to find papers that did accomplish that although I also don't see why it shouldn't work...
And if yes, how did you make the SPAGEDI input file??
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I'm currently trying to genotype around 60 brains in my lab and I'm hitting a snag in extracting the DNA. I have had no issues with DNA extraction on our newer cases, however, on our old cases that have been sitting in fixative for up to 15 years I can't even get the sample to lysis. Has anyone had this issue before or have any ideas on how to get the sample to lysis? I've tried extending the lysing time as well as muddling the tissue before adding the lysis buffer (from the qiagen micro kit).
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Please go through one of my research which is attached below. The methodology explained in the paper is appropriate for the kind of situation you are in.
Hope this helps you!
Best!
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Need the suggestion and help in Tetra ARMS-PCR standardization ... Thanks in advance.
1.What are the optimal/best conditions and reagent concentration?
2.Which one we should standardize first? Tm or denaturation/extension---Time or Temp.
3.IF we dont have positive sample how we go ahead?
4.IF alternate alleles are more than one and we are able to design the primers for it how best T-ARMS works?
We designed our primer using https://snp.biotech.edu.lk/
Problems we faced
1. We are able to get Outer and wild allele bands but not the variant allele band
2. In negative we are getting primer dimer along with another 50bp dimer/band With One SNP
3. Non specific bands
We Tried with
1. Gradient PCR for tm
2. Different primer concentration Outer Vs Inner Primer (1:10, 1:20)
3.We also tryed with increasing the variant allele primer conc.
4. Used different time and temperatures
Tm are
SNP1 OF-72* OR-74* IF-73* IR-73*; outer band 275bp, Wild allele 179bp and Variant allele 152bp
SNP2 OF-69* OR-69* IF-69* IR-69*; outer band 361bp, Wild allele 218bp and Variant allele 195bp
We used PCR conditions
94/95*c-----2 to 3 min.
95*c-----40 to 60 sec.------/
55 to 68*c-----20 to 30 sec / 20-35 cycles
72*c-----40 to 60 sec-------/
72*c-----3/5/10 min
Reaction concentrations
OF---- 10 to 20 pm
OR---- 10 to 20 pm
IF---- 10 to 100 pm
OF---- 10 to 100 pm
master mix (2X) 6-8 micro lit. Emarold master mix (takara)
Thank you..
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I did genotyping for a 92 patients, then I calculated the allele frequency for them. I get chi2 = 29, which is high, I have to reject the null hypothesis and indicate that my population is out of Hardy-Weinberg equilibrium. Is this is normal, if it is not, what I can do?
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Dear Suzanne,
If you are confident about your statistics, one or more assumptions behind the Hardy-Weinberg equilibrium must be false. It is most likely that your sampling of patients is not random and does not reflect the real population structure that they belong to.
There are two interesting possibilities.
First, their might be something unique about your group of patients which is linked to the alleles you have been investigating; i.e., the patients, because they all have the same type of medical condition may mean that the alleles you measured (which somehow underlies the medical condition), will be in disequilibrium. You could test this (perhaps) by looking at a second set of alleles that have nothing to do with the medical condition, and these we would expect to be in equilibrium.
Second, all of your patients may be from a population of people in which the alleles you are investigating are in disequilibrium (and the alleles are not directly linked to the medical condition they may all have). This might be the case if all of the patients were in a regional hospital serving an area which had little migration and mixing, but less likely if they were in a metropolitan hospital that serves a diverse city. You could test this by looking at other alleles not linked to the medical condition, and in this case we would expect them to be in disequilibrium too.
Regards, Andrew
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I am about to use sequence a set of genes in human DNA samples using ion torrent and Illumina to find variants. Do I need a positive control ? to make sure that the sequences were sequenced correctly. Do I need a negative control?
By positive control I mean using Human CEPH Genomic DNA Control (thermofisher) for example
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As far I know, if you are sequencing, the most probable situation is that, or you know predicted sequences or you know sequences of the same genes in other model animals. So the positive control does not have sense to me in sequencing a gen. And the negative either. Different thing could be if you know the sequence and you would like to confirm or demonstrate the presence of the gen in different tissues. In this situation I would use specific primers and Real Time PCR, and positive and negative theoretical controls of tissues in whcho you have some data to assume thta the gen is there and not. But this is not sequencing.
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Hi I am trying to amplify T. gondii positive DNA samples using multiplex nested PCR. I am planning to sequence the positive samples and perform genotyping. I am following this protocol as mentioned in this paper with slight modifications
In my PCR I am only using these 5 markers > L358, c22-8, 5SAG2, 3SAG2, GRA6
The multiplex PCR reaction is carried out in a vol. of 25ml containing 1XPCR buffer, 2 mM MgCl2, 200mM each of the dNTPs, 0.15mM each of the external forward and reverse primers, 1 unit of FastStart DNA polymerase (Roche) and 1.5ml of DNA samples.
The reaction mixture is treated at 95 C for 4 min, followed by 30 cycles of 94 C for 30 sec, 55 C for 1 min and 72 C for 2 min.
Multiplex PCR amplified products are diluted (1: 1) by adding 25ml of nuclease-free water. The nested PCR reaction is carried out in a vol. of 25ml containing 1XPCR buffer, 2 mM MgCl2, 200mM each of the dNTPs, 0.30mM each of internal forward and reverse primers, 1 unit FastStart DNA polymerase and1.5ml of diluted multiplex PCR products.
The reaction mixture is treated at 95 C for 4 min, followed by 35 cycles of 94 C for 30 sec, 60 C for 1 min and 72 C for 1.5 min.
I ran the gel for the PCR products following each PCR (after both multiplex and nested) and I didn't see any bands at all.
Initially, I thought qPCR positive DNA samples may have very less amount of Toxoplasma DNA to be amplified so I have run the Multiplex nested PCR again with Toxoplasma DNA extracted from T. gondii tachyzoites cultures. But I didn't see any bands at all.
I am very sure I didn't miss any reagents as I did the same experiment 3 times very carefully. But had no success.
Can anyone please suggest what could have gone wrong or any suggestions to overcome this issue?
Thank you very much.!!
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Thank you for that clarification Tharaka Liyanage and at least it shows that there is no dna degradation in the gel running buffer.
The reason for diluting the first round pcr product is partly to minimise non specific bands and partly to dilute the first round primers to avoid getting more non specific primers and mainly to avoid huge over amplification. You have to remember that 10 cycles of pcr is 1000 times more product and 20 cycles is 1000000 times more product .You have run 35 cycles of first round prc then 30 rounds of second pcr .At these levels of amplification you can get so much pcr product that it self anneals and you get long smears of amplified product but no single specific band.
About running first round product it is usually because you only use nested pcr when there is almost no dna in the original sample so there is often nothing to see after the first round. If you could see it then possibly there would be no point in running the second round but cleaning up the amplification of the first round with higher annealing temperature or adding dmso might be good enough to get a good product. Remember that while you might be adding 50ng of dna in the first round pcr almost all of this is useless host dna and very little is Gondii infection dna so there are very few targets for specific amplification with your primers
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I performed PCR yesterday afternoon and let it run over night on setting at 6 C in the thermocycler. This morning I prepared my 2% agarose gel and after it set, I placed it in the electrophoresis chamber and loaded 10ul of the samples into the wells. The volts used were 104-105v for about an hour and a half. When I took it to the imager, I got these faint bands showing. I just would like to know where in the process did I go wrong.
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Run an assay with 2 samples which have no dna in them and one normal dna sample.If the negative controls amplify bands of the right size then you have contamination
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hi all ,
I am doing a study using TaqMan genotyping assay for 2 SNPs ..
one SNP have good and clear genotyping results ,,
while the other SNP testing resulted in indeterminate results so genotypes are not plotted in the allelic discrimination plot ..
can I determine the result from the multicomponent plot using the FAM and the VIC signals ? is it acceptable ?
another question ..
I don't have a positive control ,, so my samples are run with out positive control to compare but I use an old sample repeatedly in all runs ,, but it is not set as a positive control in the instrument
in this case , depending on what parameter the instrument will calculate the threshold ?
thank you
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I still want to know how to know the result from the excel sheet . whats parmeters should i look for
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I have used ethidium bromide solution for genotyping different mouse strains successfully, but the laboratory that I am working on currently does not allow it.
Currently, I have been using a GelRed, but my PCR bands are very weak in the gel. I can see (barely) that there is a lot of DNA and I believe that the PCR reaction should be working because of that. So, maybe is something in the gel step.
I'm doing the PCR with GoTaq G2 Green Master Mix, as I used to do before. The primers are the same as the previous. Besides, I can't see the ladder.
Does anybody have any suggestions?
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Use SYBR Safe DNA Gel stain (Life technologies S33102). This is less hazardous than Ethidium Bromide and can be examined under blue light/UV. We have used it in our laboratory.
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We're looking for a working PCR protocol to genotype Il17a-Cre mice (Hirota, Nat Immunol, 2011). We're facing a few issues with the protocol recommended on the Jackson website and would be grateful for a protocol that has been tried and worked.
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Hi! I wanted to see if you ever got a working protocol for IL17a-Cre genotyping? I am having problems with the Jackson protocol as well.
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I have two arabidopsis mutants of SALK and Wisconsin Lox. I genotyped them with LP-RP primers and the LBB and P745 primers to check the homozygous lines. When I've isolated the RNA from it and used the cDNA to run a PCR with the same LP RP primers, I'm getting an amplification of two bands in wild type columbia and a single band in all the TDNA mutants. Can anyone help me figuring out the results?
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Amy Klocko Thank you for the tip! Highly appreciate it.
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I am trying to use this software to perform GEI, I am unable to perform the analysis "Some errors" is always being shown, please help me.
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Can it analyse eberhart and Russell model and how the significance of regression coefficient is calculated using GEA-R
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Hello. I'm currently dealing with a genotyping issue. Few weeks ago I genotyped a couple of mice supposed to be homozygous for a transgene and the gel showed to me multiple bands, typical of the heterozygous. I've repeated the protocol multiple times, trying to figure out the reason of the unexpected results. No changes. Due to my doubts, I've ordered another couple of homozygous mice and genotyped them again together with the previous ones and the proper wt and negative control. Surprisingly, this time all the mice appeared to be homo (just the mutant band was present). The ONLY thing that I've changed was the TAE buffer (I've prepared a fresh one and, since the one in the chamber looked dirty, with many gel residues and it was never changed since I arrived in this new lab in March, but simply refilled at every run, I discarded the old buffer, washed the chamber and fill it with the fresh TAE). Could this have made the different? I'm going crazy because I do not have any other explanation. Thank you in advance for your support.
Adele
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Yes, overused TAE can contaminate the quality of your expected bands. It is recommended you keep your chamber clean all the time especially when you are working with different/new sets of samples.
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Hey everyone,
currently doing some research on Vitamin D receptor polymorphisms, where genotyping is done mostly by RFLP. The problem is, some papers only state for example Fok1 (rs2228570) F>f as a variant. But Fok1 could be A>C / A>G / A>T accoding to dbSNP (https://www.ncbi.nlm.nih.gov/snp/rs2228570).
Lets take this paper from Mukthar et al. (2017) as an example (hps://doi.org/10.1155/2017/4171254)
"FokΙ digestion (SNP C/T) in exon 2: Restriction site presence is designated by “f,” and absence is designated by “F.” ff (CC), for example, a 196 bp and 69 bp; Ff (CT), for example, 265 bp, 196 bp, and 69 bp, bands; FF (TT), for example, 196 bp and 69 bp bands."
forward primer: 5′-AGCTGGCCCTGGCACTGACTCTGGCTCT-3′
reverse primer: 3′-ATGGAAACACCTTGCTTCTTCTCCCTC-5′
this one does state FOK-­I (rs10735810) (C/T) but the RFLP base pair length doesn't add up with Mukthar et al. (2017).
"Agarose gel electrophoresis (2%) of PCR–RFLP technique of the amplified Fok-­I genotypes. Lanes 1, 5, 6, and 7 showed heterozygous pattern (FF) genotype, where F allele at 265 bp. Lane 2 showed (Ff) genotype at 265, 196, and 69 bp"
forward primer: 5′-­AGCTGGCCCTGGCACTGACTCTTGCTCT-­3′
reverse primer: 5′-­ATGGAAACACCTTGCTTCTTCTCCCTC-­3′
Is there any way i can accurately tell the corresponding Allele to Fok1 F and Fok1 f from RFLP and primer combination?
There are other papers that just state Fok1F>f or Bsm1 B>b without any other remarks on genotype. Is there any way to accurately tell the exact variant from RFLP terminology?
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The project's budget is 12,000$ does not include buying any equipments except (for example) a genotyping analysis kit, I did a project for analyzing genetic diversity and selection signatures in four endangered cattle breeds using Illumina BovineHD kit but it was not satisfying, any suggestions? it is very important and crucial for my career.
Thanks in advance,
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You can also choose to study the diversity, evolutionary phylogenetics or domestication of a species. Large stractural varitions on genetic disease is also a choice.
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I have SSR data for 32 genotypes. In some case getting more no of allele for the markers. even hetro also coming . Now How to create input file for these in Darwin software.
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The following is the illustration on input file format for multiple alleles for microsatellite(SSR) loci in diploids :
SSR1A SSR1B SSR1C SSR2A SSR2B........... so on
Genotype-1 1 0 0 0 1
Genotype-2 1 0 1 1 1
.
.
.
So on
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Please, do some body know the composition of the genotyping TaqMan Master Mix? Thank you.
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I am really sorry to say that it is totally proprietary. I will not be able to help you beyond this.
Regards.
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this question concerns with genetic diversity and genotyping of plant materials of Boswellia sp. collected from Darfur region to configure its diversity.
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hi Singh
I hope that enough information to answer
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Why my genotyping results for a SNP is T>C, but the NCBI reported is A>G? How should I describe this issue in the article?
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NCBI provides us information of a particular loci in any one strand (i.e., either forward or reverse). To elaborate, for example you have a variant change from T/C based on your sequence retrieved from NCBI/Ensembl (the sequence is given in reverse strand), but when you search for the variant in NCBI it shows A/G (forward strand). Both are correct highlighting the complimentarity. Minor and major allele remains the same. Yes. As previously mentioned you can refer to it as reference allele/mutant allele.
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Hi all,
I have a new strain of EZH2 floxed mice that I need to genotype because I will create KO mice later, no one in our lab deals with it so there is no program for it in our thermocyclers.
This is the link in JAX website:
JAX protocol for genotyping has the cycling steps and temperatures, but they don't provide the timing for each cycle. They said that it should be optimized for your lab and reagents so they don't provide a specific protocol. I programmed a new program with the default timing in our thermocycler and it didn't work.
Attached is the protocol from JAX website, and I used these calculations to create my master mix, for every PCR tube:
12.5 ul of Green Mix (GoTaq® Green Master Mix, 2X),
9.5 ul of nuclease free water,
0.5 ul of F primer,
0.5 ul of R primer,
2 ul of DNA from my tail snips --> total volume per tube is 25 ul.
I didn't get any bands or very weak bands, the picture attached should be all positive, the first well is my NTC and the second one is my negative WT control. I checked my DNA concentrations in the Tail snips with the NanoDrop and they all had good concentration and absorbance.
My guess is that it's the PCR step that is the problem, any idea how can I find information and fix that? Especially the timing of each cycle and how to set it up.
Thanks a lot!
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Hi Ahmad,
just to check I am reading the results correctly your A260/230 values are in blue and are around 0.3-0.4. If this is the case then something is absorbing at 230 . If you are using standard dna prep kits they use a lot of thiocyanate to act as a denaturant. If you use too much crude lysate or not enough wash solution this thiocyanate can wash off with the dna and if the 260/230 is too low ( more than 2.0 is good) then this may denature the taq enzyme in the pcr leading to zero /poor amplification. If you are using a commercial kit try loading less tail tissue or doing an extra wash step before eluting your dna
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I have used PCR-RFLP for the genotyping of fowl adenoviruses previously but now I am interested in the genotyping of FAdVs by real time PCR. Kindly share your experience if someone has already used this way. Thank you
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This is a good source of primer/probe for detection of FAdVs by rRT-PCR:
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In case the normal sequence of a gene which is existed in the gene bank contains a G, but the wild type of the gene contain an A instead, does this mean that the mutant allele becomes the wild type allele ? Can this occur ? If yes. then how much time this needs?
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Kindly check the attached file
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Hello everyone,
I am looking to generate new primers for my mutant mouse model genotyping. Unfortunately I can not manage to find the FASTA sequence of the mutated genes.
I have the MGI ID of these mutant genes (usually Floxed genes) and I would like to download the FASTA sequence to design new primers, more specific ones, or one that would allow me to multiplex the PCR.
Here is for example one MGI ID (MGI:3527143 / Tnftm1.1Sned)
If anyone can help me find the information I would be much grateful.
Many thanks in advance,
Best regards
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Brian Thomas Foley I was afraid of that answer but it seems indeed that's the only option.
Thanks for helping :)
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I was wondering how one would come about finding the genotyping protocol of a specific strain of mice: C57BL/6-Il17atm1Bcgen/J x IL22promTdtomato BAC transgenic reporter. I've seen the mice strain referenced in a few papers, but have yet to find out how to get the genotyping protocol. Any help would be appreciated.
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Hi Gabriel,
one of mice that you are interested in is available from the Jackson Laboratory and they typically provide the genotyping information on their website under the technical support section of the specific mice.
It looks like you will have to perform two separate PCRs. The PCR information for C57BL/6-Il17atm1Bcgen can be found here (see technical support section):
For your other mouse strain (IL22promTdtomato BAC) you can easily check the original paper:
Generally it is good to check if your mice are available at the Jackson Laboratory. They typically provide the needed PCR information. If they can't be found there it is always best to look up the original paper that established the mouse model.
Patrick
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I have with me diploid data(genotypes)which is microsatellite markers.Can you suggest any software to produce the same fig,with pie chart as attached?
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For minimum spanning tree, you can use EdeNetworks.
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I am wondering if it is possible to use AMMI model for the analysis of the stability of 30 genotypes that were tested at seven environments?
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yes, you can do it. you can choose with Genstat software too.
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Hello. I'm a beginner in bioinformatics.
I'm handling Illumina SNP microarray data and got genotyping results of several SNPs. But in some SNP sites, for example, in the case of the SNP site of one individual is "BICF2P1141058", the genotyping result is [A/T] and the other SNP site "BICF2G630601486", the genotyping result is [T/A].
I really do not know the difference between [A/T] and [T/A]. For getting more information, I searched the Illumina SNP genotyping technical note (https://www.illumina.com/documents/products/technotes/technote_topbot.pdf) and found some information that to provide accurate SNP strand and orientation, Illumina offers strand information based on SNP genotype and nucleotide sequences surrounding the SNP.
However, I still do not clearly know why the strand information is needed. The SNP genotype result is already determined by a specific nucleotide pair. Then what kind of additional information does the strand information provide? In the case of my example [A/T] and [T/A], aren't the SNP sites just composed of A and T? Could you please tell me how considering stand information helps in the interpretation of SNP array data?
Thank you!
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The reason is that "+" strand (sense) is considered as the default DNA strand in the most cases, whereas targets for probes for SNP array can be located on any strand, according to convenience for array design. So, you need to take into account strand information in order to avoid mistakes in interpretation of data.
Also, it seems like you confuse complementary base pairs with a couple of allelic variants. In your example ([A/T] and [T/A]) both bases are allelic variants on the same strand (check if the workflow included strand conversion). Their order in the genotype record depends on what allele is considered to be the reference, and what is the alternative. This information is contained in the manifest file of corresponding microarray.
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Please explain me if the above question is true.
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Dear Nivedita Shettigar In principle, variance cannot be negative. However, we may observe 'negative' components of phenotypic variance due to uncontrollable experimental error. Under such a situation, the most reasonable value of negative estimates is taken as zero. Because of such an error, you may find the value of genotypic correlation exceeding one in either direction.
Best regards, AKC
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how work? Evaluation of the performance of this technique and its application in the diagnosis of rare blood groups
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A question on mouse crossing: Do you think it is possible to breed two genetic mutations (6kb apart) at a homologous locus onto a same chromosome, or in cis? Because the crossover between two mutations may require meiosis recombination, I thus plan to interbreed heterozygous A-b/a-B littermates, and genotyping all pups with long range PCR. Do you think this is a feasible plan? How many breeding pairs / scale would you recommend? Thanks!
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It's genetically possible, but you'd have to ask a mouse person if it's practical.
6 kb is a rather short genetic distance. Looks like on average there are 2 kb per centiMorgan. http://www.informatics.jax.org/silver/frames/frame5-1.shtml
But that can very a LOT depending on exact location, mouse strain, etc.
So, if you are fortunate that this is about 3 cMorgans in distance, then you might get 3 recombinant offspring in 100. I'd plan on genotyping more than that - 300? 500?
Better ask a mouse expert how many breeding pairs that would take. And see if the recombination rate for your particular locus and mouse strain are known.
Good luck!
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Where can I find the sequence for mGFP6? better yet, what primers do you use for genotyping to check if the part of the construct containing the mGFP6 sequence is there? I was trying to imagine some sorghum tissue that was supposed to express mGFP6, but I couldn't see any fluorescence, so now I'm trying to genotype the plants to see if the construct/mGFP6 sequences are actually in the plant tissue.
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Thank you Sharif Hasan Siddiqui I found it!
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Thanks
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Hi,
I may be able to help you here.
I still have a lot of reagents for the PapType13 test (Jenkins A, Allum AG, Strand L and Aakre RK. Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus realtime PCR. J Virol Methods, 187: 345-351 (2013)) left over. We never managed to find an investor to bring the test to market and the company folded, but it's a good test and we'd like to see it used, even though we don't have any commercial interest in it any more.
Whether it is suitable for uses would depend on the instrumentation you have available. You need a AB7100, 7300, StepOne Plus, or another instrument that can detect FAM, VIC, NED and ROX simultaneously.
DNA extraction is not part of the kit, so I can't help you there, though I could probably come up with some suggestions that won't cost too much. One of the QIAGEN genomic DNA kits would probably work well. Or you could just do an old-fashioned proteinase K - SDS - Phenol/Chloroform extraction.
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Thanks
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Oscar L. Sierra Thank you very much for helping me in showing the direction and I have sent my request to Dr. Sin Hang Lee. I hope some positive thing could happen soon. Thank you
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Dear fellow scientists,
I like to performed a direct amplification of a fragment of 222-bp DNA from human genome and use is for further analysis. By using the following methods, I obtained amplicons of the target size for a few times, and then I repeated the same experiment with the same procedure, but I only got smears. I tried to use fresh primers, fresh polymerase, and freshly prepared samples, but nothing helps. Since then I only get smears. It would be really amazing if anyone knows what might be the reason.
The method I tried was:
  1. Rub a sterile cotton swab against the inside of the cheek for 30-60 seconds.
  2. Place the swab inside a 1.5 mL tube and add 200µL de-ionized water.
  3. Close the id of the tube and then vortex for 5-10 seconds.
  4. Cook the swab + water at 95°C for 10 min to break the cells.
  5. Remove the swab and use the supposedly crude cell lysate for PCR.
  6. PCR protocol:
Component (total volume: 25 μl)
  • 10 µM Forward Primer: 0.5 µl
  • 10 µM Reverse Primer: 0.5 µl
  • crude cell lysate: 5 µL
  • OneTaq 2X Master Mix with Standard Buffer: 12.5 μl
  • Nuclease-free water: 6.5 µl
PCR program (touchdown PCR) is described in the attached picture.
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This is just the case of "TOO HIGH" DNA. Try to reduce the DNA concentration and adjust it to 20-30 ng / 25 µl reaction. I you have a nanodrop, try to take a rough reading of the DNA concentration of your cell lysate.
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Dear Colleagues,
I am new to genes and genotyping and I need help. I am trying to figure our how AA, AG, and GG genotypes map on to A1 for DRD2/ANKK1 Taq1A polymorphism(rs1800497) and Val or MET for COMT Val158Metpolymorphism (rs4680). Where can I find this information presented in a way that a gene-naïve person will understand?
Thank you very much in advance,
Monika
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Hi Monika,
For me ensembl.org is the easiest. For example COMT
1. you search for the human gene
2. Open the gene file:
3. Select one of the longest and best annotated transcripts
4. Select cDNA in the left panel
If you don't see the SNPs go to Configure the Page and select Variants. If you prefer, you can visualize the SNPs as well in the gene view.
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Genotyping data is from Illumina Global screening array 24 v 2.0 and ConsistencyDupSNP.sh file contains list of pair of duplicated SNPs in GSA24 v 2.0.
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  • Since this is a code/script file, it could not be uploaded as .sh. Please remove .txt from file before use (attached: ConsistencyDupSNP.sh). Try if it works.
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Genotyping PCR is used to determine the genotype of an organism (e.g., WT vs. mutant, or WT vs. transgenic)
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The so-called "left" and "right" border primers both are supposed to face outwards into the genomic region where the TDNA insert landed. Most folks use a gene-specific primer (F primer or R primer that you design) paired with a left border primer because the left border of the TDNA is more likely to be present due to the way the TDNA inserts into the genome.
F primer LB RB R primer
[gene of interest]<--[TDNA]-->[gene of interest]
The assumption is the the TDNA insertion is so large that you can't make a product from F to R. You use the estimated location of the TDNA from the stock center as a guide for designing these primers. General rule, see where they say the insert is, back off ~500 bp, and look for a good primer.
So, you set up PCR with F + LB; F + RB; R + LB; R + RB to see which way the insertion is facing in the gene of interest.
Let's say the F primer and LB give a good strong band in your mutant allele. The F + R give a strong band in the wild type allele.
Set up 2 PCR reactions for each plant you want to genotype.
Homozygous wildtype: band from F + R; no band F + LB
Heterozygous: band from F + R; band from F + LB
Homozygous mutant: no band from F + R; band from F + LB
Include a FULL set of controls and double-check any that you think are homozygous (PCR can fail for lots of reasons).
In reality, TDNA insertions are complicated, often contain insertions or deletions or multiple TDNAs or chromosomal translocations (or some combination of all of those things). That was the basis of my thesis.
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I am working on MD simulation project aiming potential drug discovery. I took mutants of a certain group and modelled protein structures with and without transit peptide sequences. My one aim is to see whether there is any structural, physiological or functional changes for the presence of transit peptide sequence. In Acinetobacter baumannii, with/without transit peptide ,what is the functional and structural difference occur due to mutation?
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If I understand your question correctly you wonder whether signal sequences affect folding of the mature part of precursor proteins.
It is good to realize that proteins are exported across the inner membrane by different pathways. Either by the Sec pathway or by the twin-arginine translocation (TAT) pathway. Specific Sec- and TAT-dependent signal peptides target unfolded or folded proteins, respectively. There are also some proteins lacking any apparent targeting signal by the so-called non-classical secretion pathway.
See for example:
Renuka Kudva, Kärt Denks, Patrick Kuhn, Andreas Vogt, Matthias Müller, Hans-Georg Koch, Protein translocation across the inner membrane of Gram-negative bacteria: the Sec and Tat dependent protein transport pathways, Research in Microbiology, Volume 164, Issue 6, 2013, Pages 505-534, ISSN 0923-2508, https://doi.org/10.1016/j.resmic.2013.03.016 (see enclosed file).
Indeed, there are indications that signal sequences affect the stability and folding of the precursor (or better the mature part of the precursor):
Beena, K., Udgaonkar, J. B., & Varadarajan, R. (2004). Effect of signal peptide on the stability and folding kinetics of maltose binding protein. Biochemistry, 43(12), 3608-3619.
Prajapati, R. S., Indu, S., & Varadarajan, R. (2007). Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins. Biochemistry, 46(36), 10339-10352.
Singh, P., Sharma, L., Kulothungan, S. R., Adkar, B. V., Prajapati, R. S., Ali, P. S. S., ... & Varadarajan, R. (2013). Effect of signal peptide on stability and folding of Escherichia coli thioredoxin. PloS one, 8(5), e63442.
Hope this helps you a bit further. Best regards.
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Most of my job duties revolve around mouse colony maintenance and genotyping. Our protocol for tagging and tailing is probably not dissimilar to most institutions, I take a 2-5 mm tail snip and cauterize immediately. We had four consecutive mouse deaths shortly after tailing last month and just today had two more deaths. This is puzzling, as this number of deaths has not occurred in such quick succession since I started five months ago. Is it possible for a mouse to bleed out and die from a cut that small if it somehow opens back up? There also isn’t a strain correlation between the mice that died, all were from different litters and strains. Unfortunately apparent cause of death is not listed when we get an animal death notification from the vet tech so I’m at a loss as to what’s going on here, has anyone else experienced this?
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Hi,
When I used to collect tail biopsies, we used a mild anesthesia like Isoflurane (closed chamber based anasthesia and not the injection based) to put the mouse in a sedated condition (ours was a trangenic mice colony). We then used to snip 5 mm of tail after sanitizing with alcohol and after snipping, we used to wipe the cut end with an alcohol swab till the bleeding stopped.
Here is what I can suggest:
If you are putting mice in a mild anaesthetized condition, lower the amount of anasthetic chemical used or lower the time for which the animal is kept in the chamber.
Cauterizing might be causing stress, especially, if it is a transgenic colony, you can try using alcohol swabs till the bleeding stops. It might also be due to an infection after cauterizing though that is a bit unlikely.
Please check for other factors which are not tail snipping related. Check for feed and water in the cages in case there is a death in future. You can also get the nutritional value of the feed tested if you encounter a weight loss among mice in your colony.
Further, if the mice which died, were visually thin or underweight, you can consider providing your colony with nutritional supplements like cornflakes mixed in oil etc.
Lastly, at times, male mice usually from different litters or different parents fight. So if the dead mice have visual marks of a fight etc. you can consider housing only littermates together.
Hope it helps.
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I am collecting embryos for histological analysis and need to genotype them beforehand. In the past I have worked with older mouse embryos (E8.5 and up), where it is easy to remove the yolk sac and use it for genotyping. However, I now need to start collecting E6.5 embryos. Given that they are so small, I am not sure whether there is enough material to be able to save the epiblast for histology and use other tissue for genotyping. Does anyone have experience with genotyping E6.5 embyros?
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Article Oocyte-derived Smad4 is not required for development of the ...
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Hello guys,
I need your help. I used to do PCR for genotyping of my LCK Cre mice. The PCR was working well. You can see in Fig A (Attached picture). No template NT was clean and negative control was negative. The positive band was 300bp. But as you see in Fig B my NT has 2 bands which one of which is similar to the expected positive band! I cleaned all the equipment and changed the material and repeated the PCR but still, there are bands in NT and negative control Fig c.
I really appreciate your help to figure out the problem.
Best regards
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41 cycles is a lot. Almost anything will amplify given that many cycles.
Have you tried this with 30-35 cycles? If you aren't seeing your desired band after 35 cycles, you should probably consider re-designing your primer set. Doing so may improve your signal to noise ratio.
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I am working on quantstudio 5 system with diagnostic test kit - AZF Microdeletions REALTIME PCR genotyping kit (master mix is aliquoted to every separated pcr tube, I only added taq polymerase, mineral oil and DNA for pcr preparation step)
After doing several tests, i found some abnormal amplification curves (heaved curve with a small rise at very end pcr cycles) (attachment picture).
Does anyone know what 's happened?
Besides, the threshold on the picture is set automatically by QS5 system, can I trust this threshold to determine whether my pcr target presents or not?
I am really appreciated for your help.
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What happened is that you have 0 amplification in your samples. Look at the scale on the Y axis of your plot. The data are being stretched to fit the size of the screen. That is exaggerating the fluctuations below the baseline - it's just an artifact of the way the data are presented.
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I make sure to add a DNA sample to every PCR tube. What can be the reason for the faint or no bands after Mouse Genotyping PCR?
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No amplification can be caused by a lot of common problems. You can narrow this down a lot by running control reactions: positive and negative controls for your genotype of interest, AND a known-good control primer set that should amplify if any mouse DNA is present. When I was doing genotyping I used to mix the genotyping primers and control primers (GAPDH gDNA) in a single reaction. This way two bands is positive for the transgene, one band is negative, and no bands is a reaction failure.
If control primers are working, the issue is with your genotyping primers. Either the primer set is bad (dimer problem, running at the wrong temp, a primer is around the wrong way, or something like that) OR somehow all your samples are transgene negative.
If control primers are not working, the problem is with your samples or PCR. Check the sample concentration and purity - is there any way it contains PCR inhibitors? It might need to be concentrated, diluted, or cleaned up. Check your reaction setup to make sure all reagents are included at the right concentration, and double check your thermal cycler settings. Finally, make sure that if you're using a hot start polymerase, you include the hot incubation to initialise the PCR.
Good luck! :)
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I am crossing two knock-in C. elegans strains. After mating and I started genotyping the homozygous. Right now I am at 5th and 6th generation. The PCR results showed there were 50% positive bands for the 5th generation, but for the 6th generation, there was only less than 25% left. I have tried to renew my reagent and the results stay the same. During the period, I haven't seen any lethal phenotype. Has anyone seen this happened before? Thanks.
Yuzhi
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An allele doesn't have to be lethal to be at a selective disadvantage compared to wild type. Are you finding any homozygous individuals? Or are they all segregating?
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For sex determination in nutria embryos I used SRY primers, which give a band for the males, as they should, but the primer I used for control, rps12 (S12) doesn't give a band. Are there any useful primers for nutria?
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This post is going to be a little long, so I apologize in advance for bothering you. I learned that quality control is super important so I want to make sure that I do this right.
I have several questions for my QC pipeline but hopefully nothing complex. I am just having some hard time to find the right commands. and when I do I am not sure of the inputs.  Note that I am using plink v1 (because all the examples I found use plink v1) so if you can give me the v2 version that would be helpful but it is not my main concern. Note also that I am using data from UK biobank so every chromosome is in  separate files (genotyped: .bed .bim . fam  / imputed: .bgen .mfi .sample)
My pipeline is based on 2 parts :
1- per individual filtering
      a. Removal of individuals with excess missing genotypes       b. Removal of individuals with outlying homozygosity values       c. Removal of samples showing a discordant sex       d. Removal of related or duplicate samples       e. Removal of ancestry outliers
2 - per sample filtering
      a. Removal of SNPs with excess missing genotypes       b. Removal of SNPs that deviate from Hardy-Weinberg equilibrium 3. Removal of SNPs with low minor allele frequency       c. Comparing minor allele frequency to known values
My code:
1.a: ./plink --bfile ukb_cal_chr{}_v2_reduced --missing     --out  output1 comment: use R to remove individuals with missingness >.05
1.b: ./plink --bfile ukb_cal_chr{]_v2_reduced --het             --out output2   comment: use R to remove individuals with the absolute value of F >.05
1.c: ./plink --bfile ukb_cal_chr{}_v2_reduced --check-sex  --out output3   comment: not sure what the input is ? then in the output remove individuals with status =problem
1.d: ./plink --bfile ukb_cal_chr{}_v2_reduced --genome --min 0.05 --out output4 comment: using R in the output  output4.genome, for every pair remove the one with the lowest genotyping rate (unless there is a command for that  in plink ) (is that right?)
!!! However, I found that --genome takes too much time, is there another way?
1.e: ..... comment:   I found this command : 
plink --file data --cluster --neighbour 1 5
comment: but I am not sure what it did and how to use the output to filter the individuals and what  the input file is (file or bfile)
2 - a,b,c : ./plink  --bfile input  --maf 0.01 --hwe 1e-6 --mind .1 --geno .1 --make-bed --out output
That's it for my pipeline. my main questions are related to the red parts, so just 3 questions. Also, if you found errors in my pipeline can you please correct me?
In conclusion here are my 3 questions:
- since I have one file for each chromosome, is the input of the command 1.c , the chromosome X?
- the command -- genome takes a lot of time, is there a way to speed it up or to estimate the relatedness of individuals in another way?
- I am still not sure how to filter ancestry outlier using pca?
Can you please help me? thank you
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Dear Emile Dimas
first Q:
plink --bfile data --missing-genotype N --make-bed --mind 0.05 --maf 0.05 --geno 0.1 --hwe 1e-6 --recode --out gwasclean
Your way of QC is correct.
second Q:
It is better to use GCTA and split the calculation by applying --thread commend or calculate --grm for each chromosome and finally merge them together in an individual file.
third Q:
there is a way to put a couple of first PCs according to their eigenvalues as covariates in your analysis for adjustment and lowering inflation rate.
Regards
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I am about to do an experiment of tetra ARMS PCR, I need to calculate the final annealing temperature for the four primers, how can I do it?
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I think you might need to do Gradient pcr to find a good setting.
if you use NEB polymerase, you could check this website for some initial idea.
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Hi all,
I desperately need help troubleshooting my genotyping PCR!
I have a mouse strain with a point mutation and for the last two years I have been genotyping them by first doing a PCR on the region of interest, followed by restriction enzyme digestion (the mutated sequence has a cleavage site, the WT does not). So far, so good - because now suddenly it does not work anymore.
The problems started about a year ago: for some samples I did not get a band anymore or just a smear. I tried different things, eventually designed and ordered new primers and indeed I had nice results again.
But now, 2 months ago, again the same problems: all of a sudden for about half of my new samples there is no band anymore or just very weak ones or just an unspecific band at a strange size (see attached files).
I have tried: pcr on pcr, fresh reagents, different lysis reagents for the DNA extraction, different dilutions of the samples, another genoytping PCR (this one works), new primers (same result: some samples work, some don´t). I am at the end of my wisdom and very much hoping for new ideas from the RG community. Any comment is appreciated.
Thank you in advance!
Best regards
Felicia
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You are right, I will do.
Thank you very much!
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Dear all,
May I ask whether somebody has experience in extracting DNA from glutaraldehyde fixed samples that were prepared from electron microscope images? Does such extracted DNA work for genotyping? Thanks in advance.
Best regards,
Ruping
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Interesting question. Following the discussion.
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Need help with my Genotyping. I cannot get WT bands to show up on gel.
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May I check what does WT and KO refers to ?
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Since 1999, an approach called MLST has been used as a tool for global epidemiology. Do you think that the MLST will lead us to the same destination as the gene sequencing and genomics did?
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One of the greatest advantage of using MLST is that sequence data are unambiguous and the allelic profiles of isolates can easily be compared to those available in MLST Database. With the availability of wgMLST and cgMLST, MLST provides a high discriminatory power. No Lab to Lab variations in MLST, unlike PFGE, RFLP, etc. The disadvantages of MLST includes high cost and computational requirements.
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Hello,
I am running PCR for genotyping. I didn't have problem till this Summer, when I couldn't see any band on the gel. I checked primers, Taq, etc. I bought new stocks etc. But still I can't get any result. The only thing I changed compared to before, is the Thermal Cycler machine, but everyone was telling me that it was impossible my bad results were due to a malfunctioning of the machine. I then went to check the manuals of the 2 machines and I noticed that the main difference is their ramp rate: 1 degree celsius for the machine I am using now vs 4 degrees for the previous machine. Can this affect my PCR cycles and, therefore, my genotyping results?
thank you
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Paola Salmas I would not change the ramp rate. Shame that you cannot run a gradient but pcr works very fast and works at the temperatures between the set parameters. I would assume that the annealing temperature is the main problem because too high means a failed pcr so I would just drop the annealing temperature by 1-2C and just in case the melting temperature is the problem raise that by 1C. Try also to use exactly the same tubes/strips/plates that you were using on the old machine or ,at least, use tubes that other people are succeeding with on this machine. You could also consult the equipment manual to find out what volume of pcr mix the machine is calibrated for just in case it is calibrated for ,say, 50ul and you are using 10ul reaction volumes
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I'm trying to do genotyping of mouse DNA by the use of qPCR.
I'm using HTRA1 primer and GAPDH primer and SYBR green.
My problem is that I do not see a difference between my transgenic and the wildtype? I would expect a difference in the HTRA1 gene between the transgenic and the wildtype.
Anyone with the same problem?
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every change in genome structure can have a deep impact on transcriptome answer. by transgene you mean that the HTR1 gene comes from an other species?
but first, did you test your primers to be specific to your targets? the best way to do it is on UCSC website with its in silico PCR tool. efficiency of the primers on amplification in qRT-PCR is also a landmarq for this method.
did you use the delta delta method as derived from this paper ( )?
fred
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Hi, I have two data sets from the illumina omniexpress snp array platform. The first data set was mapped using the GRCh37 build and the second one was more recently read using the GRCh38 build. Not surprisingly when I've tried to merge the files in PLINK for a larger analysis it comes up with the warning snp rs... is in a different genetic position. Is there any way to update the build of the first data set? Or suggestions for how best to proceed, I haven't done much genetic analysis before so any help would be welcome :)
Best,
Mari
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Hi,
Between GRCh37 and GRCh38 some of the items should be the same. For those that differ, I recommend the website:
Unfortunately, I'm afraid that with a large amount of data, the conversion may be a time-consuming process, but it's always a solution.
Hope this helps! Feel free to ask if you have questions about how to use the program to convert.
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I have allele frequency data of 20 SNPs generated from genotyping of a population (n=300). the region of gene under study is naturally polymorphic. I want to compare the data with global MAF data (1000 genomes project) to find statistical significance. Can this be done? if so then, which statistical analysis would be appropriate to do this?
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Ken G Dodds Thanks a lot for your recommendation.
Bharti Vyas the SNPs are not linked so, LD studies are not applicable in this case.
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I have 2 genotypes, one resistant and another susceptible with two repeats of each genotype for stress and control condition. I will analyze them with one concentration of heavy metal  at two time points in comparison with control genotypes.  Briefly two genotypes, two time points, two repeats for heavy metal treatment and control, one heavy metal concentration. Totally 16 samples. could any one give aa advice? Regards
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YOU CAN TRY NETWORK ANALYST SOFTWARE FOR DIFFERENTIAL EXPRESSION ANALYSIS ,
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I recently acquired some Genotyping by Sequencing (GBS) data for my study group (a plant in the legume family).
I would like to blast my data against the closest possible organism with a reference/draft genome to identify which regions are under selection.
How do I find out which is the closest related organism with an available genome to blast against?
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We have been trying to run genotyping protocols. We ran a gradient PCR to re-optimize the annealing temperature, tried to change the ramp speed and also modified the DNA purification protocol. Has anyone faced this issue? Would appreciate any advise on how to get this working!
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go conservative on all parts of the cycle until things are working....lower annealing, higher denaturation and longer times. Your gradient tells you that the annealing temperature is not the problem so more likely it is the denaturation temperature has changed. Run pcrs with other primer sets and with reagents that do work on another machine just in case the regents/primers have degraded and use dna that has amplified before. Are you sure that the machine is cycling?
Try a cycle of 60c then 4c and run it touching the block and see if it feels hot/cold at the appropriate times. Check the expected total cycling times eg if the pcr 30 cycles should take 2 hours then you have a block problem if it takes 4 hours.
Read the instruction manual especially the programming and troubleshooting sections
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Hello
I am studying genotyping by RAD-seq.
I have interests in population genetic structure within wild plant species.
So I would like to use GATK for genotyping / variant calling.
However,
I felt in a problem.
My working sentence is
"java -jar gatk-package-4.1.2.0-local.jar HaplotypeCaller -R /mnt/c/Popgen -I /mnt/c/Popgen/samtools-1.9/sample18.bam -O /mnt/c/Popgen/GATK/sample18.vcf"
and then,
I got this result.
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11:45:13.056 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/mnt/c/popgen/GATK/gatk-package-4.1.2.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
May 07, 2020 11:45:14 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
11:45:14.793 INFO HaplotypeCaller - ------------------------------------------------------------
11:45:14.793 INFO HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.1.2.0
11:45:14.794 INFO HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
11:45:14.795 INFO HaplotypeCaller - Executing as bwook1243@bwook1243 on Linux v4.4.0-18362-Microsoft amd64
11:45:14.795 INFO HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_242-8u242-b08-0ubuntu3~18.04-b08
11:45:14.796 INFO HaplotypeCaller - Start Date/Time: May 7, 2020 11:45:13 AM KST
11:45:14.797 INFO HaplotypeCaller - ------------------------------------------------------------
11:45:14.798 INFO HaplotypeCaller - ------------------------------------------------------------
11:45:14.799 INFO HaplotypeCaller - HTSJDK Version: 2.19.0
11:45:14.799 INFO HaplotypeCaller - Picard Version: 2.19.0
11:45:14.800 INFO HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
11:45:14.801 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
11:45:14.801 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
11:45:14.802 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
11:45:14.804 INFO HaplotypeCaller - Deflater: IntelDeflater
11:45:14.808 INFO HaplotypeCaller - Inflater: IntelInflater
11:45:14.809 INFO HaplotypeCaller - GCS max retries/reopens: 20
11:45:14.809 INFO HaplotypeCaller - Requester pays: disabled
11:45:14.810 INFO HaplotypeCaller - Initializing engine
11:45:14.815 INFO HaplotypeCaller - Shutting down engine
[May 7, 2020 11:45:14 AM KST] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 0.03 minutes.
Runtime.totalMemory()=288882688
java.lang.IllegalArgumentException: File is not a supported reference file type: /mnt/c/Popgen
at htsjdk.samtools.reference.ReferenceSequenceFileFactory.lambda$getFastaExtension$0(ReferenceSequenceFileFactory.java:252)
at java.util.Optional.orElseGet(Optional.java:267)
at htsjdk.samtools.reference.ReferenceSequenceFileFactory.getFastaExtension(ReferenceSequenceFileFactory.java:252)
at htsjdk.samtools.reference.ReferenceSequenceFileFactory.getDefaultDictionaryForReferenceSequence(ReferenceSequenceFileFactory.java:222)
at org.broadinstitute.hellbender.utils.fasta.CachingIndexedFastaSequenceFile.checkFastaPath(CachingIndexedFastaSequenceFile.java:166)
at org.broadinstitute.hellbender.utils.fasta.CachingIndexedFastaSequenceFile.<init>(CachingIndexedFastaSequenceFile.java:129)
at org.broadinstitute.hellbender.utils.fasta.CachingIndexedFastaSequenceFile.<init>(CachingIndexedFastaSequenceFile.java:111)
at org.broadinstitute.hellbender.utils.fasta.CachingIndexedFastaSequenceFile.<init>(CachingIndexedFastaSequenceFile.java:96)
at org.broadinstitute.hellbender.engine.ReferenceFileSource.<init>(ReferenceFileSource.java:35)
at org.broadinstitute.hellbender.engine.ReferenceDataSource.of(ReferenceDataSource.java:27)
at org.broadinstitute.hellbender.engine.GATKTool.initializeReference(GATKTool.java:423)
at org.broadinstitute.hellbender.engine.GATKTool.onStartup(GATKTool.java:693)
at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.onStartup(AssemblyRegionWalker.java:161)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:137)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:191)
at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)
at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:162)
at org.broadinstitute.hellbender.Main.mainEntry(Main.java:205)
at org.broadinstitute.hellbender.Main.main(Main.java:291)
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I think that the connection to Google compute engine is not working on my computer. Isn't it?
I prepared reference files (fasta (*.fa), *fa.fai, *.dict).
How can I solve this problem?
Please give your kind help.
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Hi,
There would be a problem with JAVA.
GATK does not support the latest releases of JRE or JDK.
Switching to version eight might solve the problem.
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Ammi Analysis or GGE Biplot Analysis ?
I have several Nitrogen application treatments in a three year Winter wheat Experiment.
Can I consider each treatment as an environment and run an AMMI GGE Biplot Analysis to look for the performant and stable genotype in each environment?
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both are suitable
but have seen any change in any of genotype after the year passing?
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Hello,
I currently setting up an experiment to study the role of a set of genes that span 150 Kilobases in my cell line. So I plan on generating a 150 KB deletion by nucleofecting Cas9 with 2 sgRNAs into my cell line. What is the best PCR genotyping strategy to validate my deletion? I was planning on doing three separate PCR reactions (attached in the figure, primers sets are indicated by different colors).
For anyone that has experience doing this, is this strategy okay or are there better PCR strategies to validate a deletion of this size?
Thank you, I really appreciate all the help.
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Remember, you can only find what you are looking for. Paul is correct that you also need include a primer set inside the deletion area and primer set with one inside and one outside. Ideally, you could sequence the "boundaries" of the deletion and design a new primer that will only make a product when the deletion is present.
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I' m going to analyze Transcriptional profiling of some drought related genes among field selected chickpea drought tolerant genotypes under contolled condition by RT-PCR and Real-time RT-pcr, but I don't know how and on what basis I should choose these genes ( two or three genes I want to select). please guide me to what genes should I select ??
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Zhao & Fernald (2005) have developed a tool that calculates qPCR efficiency from the kinetics of each individual reaction directly, without the need of a std curve.
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Hi, should we have triplicates for standard in qPCR? Or not necessary?
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Hi,
I am genotyping newly constructed trangenic mice knocking down target gene. I have got F1 generation from F0. And DNA examination indicates that the target DNA fragment has been excised. The bugs are that PCR targeting mRNA and WB show that the target mRNA and protein are not knocked down. PS: One of my mRNA primers locates in excised region. The only reason I think of is the PCR contamination and bad behavior of first antibody as DNA has been excised. Could you pls help me with this problems? THANK YOU.
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Run a full set of controls with your experiment. It's entirely possible that your mice do not have the transgenic knockdown at all. Better to learn that now with PCR than to waste months or years using a non-useful mouse line.
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Hello,
I am using a mice line which is a cross product of Sun1-tagged mice (Jax #021039) with VIP-Cre mice (Jax # 010908).
I'm trying to establish a PCR genotyping protocol to detect whether an unintended recombination occurred at some point during the embryonic development.
Since I couldn't find a map for the KI sequence in the Sun1-tagged mouse - the process of planning the primers for PCR genotyping is a bit tricky.
I would appreciate if someone that has performed this type of genotyping for the ROSA26-pCAG-LSL allele could help me with this.
Alternatively, it would be also very helpful if someone could refer me to where I can find a map for Jax #021039 mice. I couldn't find it through MGI (or perhaps I missed it).
Many thanks!
Eran
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Unfortunately, this information is not enough; they also refer to the paper that this mouse line was generated for - but I couldn't find the actual sequence.
I've tried to genotype this with primers that I planned based on the information in the paper (see reference below) - but it didn't work for one condition and for the other one I got a PCR product also in the negative control.
Therefore I must have the explicit sequence or a specific primers that someone already tried successfully.
Anyhow, thank you so much Sheng Sun for all the effort! I really appreciate this :)
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We created parent mice with long trinucleotide repeats. We did genotyping on offspring mice and it worked well. But the most recent generation showed no band at all. We checked the genotype of parents mice and nothing's wrong. Why this generation were all negative? Could that be related with the age of parent mice? All protocals used were unchanged.
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Gene instability in offsprings may be related to the genetic recombination during the generation of sperms and OVA. However, it is also a random event which should not cause a uniform deletion of a particular gene in all the offsprings.
You should investigate your genotyping further.
Most probably, the inactivation of proteinase-K in lysis buffer, post digestion of tissues.
Complete inactivation of proteinase-K occurs at temepratures over 85 degree centigrade with incubation over 2 hours.
If for any reason the proteinase-K remains active, it will cause degradation of the DNA polymerase used for PCR for genetyping.
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Using the targeted method “Allegro genotyping”, I want to genotype natural accessions from different geographic regions of the wild strawberry Fragaria vesca. I need a SNP array containing at least 5000 SNPs, but I can’t find one specific for this species. Any of you knows where I can find this data?
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Hi Vit,
Sorry I know every little about the array thins.
But I find this species has a well established reference genome with a small genome size (~240Mb). How about using WGS instead?https://www.rosaceae.org/analysis/252
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I am trying to do a cross using daf-16(m26) strain but the problem is to genotyping of this worm.
do anyone have a primer seq for the specific to the daf-16(m26)??
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