Genotyping FAQs

What is the standard quality and quantity of DNA for iPLEX or hME Genotyping reactions?

• We recommend an A260/A280 ration of 1.7 – 2.0 and 5 – 10 ng/ul of DNA per reaction well.

What is the upper limit of DNA quantity for iPLEX and iPLEX Gold reactions?

• We have found that up to 50ng the reactions work fine, then performance starts to degrade beyond 50ng for primer extension reactions.

How do you recommend quantitating the DNA for our Genotyping reactions?

• We recommend either UV spec or Picogreen to quantitate the DNA.

What is the maximum plex level per well for iPLEX Gold?

• The maximum plex level for iPLEX Gold is a 40 plex and your MALDI-TOF needs to be tuned for iPLEX Gold.

Where do I download the new Assay Designer for iPLEX Gold?

• You should go to www.sequenom.com and visit the iPLEX Gold link.  At the top of the page you will see an ….Upgrade to iPLEX Gold link and if you follow that link you can download the Assay Designer rev 2.  You will need a realSNP account get into the download portal to download the software.  The download portal is case sensitive for both your email address and your password.

How do I get a My SEQUENOM account?

• Please visit MySEQUENOM.com and register online.

Do we have a standard recommended protocol for customers doing low multiplex (1-18) iPLEX reactions?

• We have come up with a low plex protocol for iPLEX that uses half the amount of termination mix and half the amount of iPLEX enzyme that is required for standard iPLEX reactions of 19 – 40 plexes.  Please check the iPLEX Gold Application guide for specific protocol reactions.

Can you use WGA DNA for iPLEX and iPLEX Gold?

• Yes, you can use WGA DNA for both iPLEX and iPLEX Gold.  You must have high quality starting DNA and realize that your clusters may not be at tight as you would observe with gDNA.

Is extension primer optimization really important for my iPLEX reactions?

• Yes!  As oligo of higher molecular weights fly slower than the lower molecular weight oligos, you need to ensure that your extension primers of are as equal intensity as possible.  The rule of thumb is that your lowest intensity peak is at least 50% of your highest intensity peak.  You can choose to do a 2-step, 3-step, 4-step or linear regression to optimize your primers.  The primer optimization method is located in Appendix A of the iPLEX Gold Application Guide.

Is doing a volume check prior to spotting my plates an important step in the overall Genotyping process?

• Yes!  The chip is what bridges the gap between the reaction plate and the mass spectrometric analysis.  If material is not properly spotted on the chip, you will have bad results.  Make sure your liquid level is the same for all wells (no evaporation problem).  Make sure your pins have been conditioned properly (daily and weekly maintenance).  Check the resin in the well to make sure that it is all at the bottom of the well.  You can find how to perform a volume check in your nanodispenser’s manual.  The volume range for good results is 15-40 nL.  An average volume of 25 nL is a good target to shoot for.  If you observe high pin to pin variability during volume check, clean and condition your pins.

Plate Editor in Typer 3.4 does not properly apply sample groups.

• The problem with plate editor v3.4 sample list improperly applying samples with certain sample groups has been determined.  This problem has been identified in that a sql statement did not order the samples being queried from the database.  Most of the time the order of the sample list is sequential (1,2,3,4,5,6,7,8,xxx) in the database.  However there are some sample lists that are not sequential (1,3,5,4,2,8,xxx) which is perfectly fine, but the sql statement must take that in account. 
  
  Solution – refer to PSB-14-Typer 3.4 which tells you to replace the old PlateEditorSQLStatements.txt file with a new PlateEditorSQLStatements.txt.

Triallelic SNPs do not properly import in Typer 3.3.x.

• This problem was observed and has been corrected in Typer 3.4.  The only work around for this problem in 3.3 is to import all assays as bi-allelic assays and then manually edit them in Assay Editor.  This problem seemed to be confined to the importing of assays only.
• Typer 4.0 allows the cluster plot to display graphical information about tri- and tetra allelic assays (which is not available in Typer 3.4).  This software will allow you to query any two alleles in the reaction at one time and you may switch which two alleles you are looking at.  You cannot visualize 3 or 4 alleles at the same time however.

Assay Editor Genotype Naming bug.

• We have identified a bug in Assay Editor (Typer 3.4) that you may encounter if you try to edit a Genotype Call. This is a relatively trivial bug, but may cause confusion if you want to change an analyte name.  
  
  Here is an example: Under the “Edit Assay” Tab you may want to change the Genotype Calls. You can go ahead and click on the diagram, edit the Analyte / Call to the desired values and then hit save.
  
  Here I changed the G to a C and the T to an A and saved, but the diagram still shows the original Analyte / Calls in addition to the new ones.  The bug is that this diagram does not reflect the newly changed Analyte / Call.
  
  In reality the new Analyte / Calls are saved to the database and are reflected under the “Details” tab.  The “Details” tab (below) is where you can check to see exactly what values are currently in the database. 
  
  Solution – This was addressed in Typer 4.0 and corrected.

Can you use a 10 kb amplicon for an hME/iPLEX reaction and skip PCR for multiple SNPs?

• Using a 10 kb amplicon as a starting material without PCR amplification for a hME reaction should be feasible.  We worked with doing more genomic amplification studies before at Sequenom.  One drawback which I will point out to you (as R&D pointed it out to me) is that they noticed a lot of false positives in when doing genomic amplification which is one of the main reasons why we use short amplicons in our present protocols.  Your extend primer could anneal on a site which is not the target region and then become extended.  Further, there is no quick and easy control experiment for these false binding sites.  If you expected a C or T and got an A then you have this problem going on.  Just to give you some warning of possible pitfalls you could encounter.
  
  As for the quantity involved, based on your system, R&D has suggested 1 ug of 10 kb DNA template to be used for the extension reaction which is pretty close to my 2.7 ug estimate I made. This number should be accomplishable where you have 200 ng/uL stock solutions but a bit painful perhaps where you’ll be using more than you wanted to.   I strongly recommend an exploratory reaction series to test if this will work.  There are two factors to worry about 1) concentration (you might be able to get away with using less) and 2) false positives from the extend primers sticking somewhere you don’t want them to on your 10 kb stretch.
  
  
  Using higher quantities of template DNA can sometimes solve the problem of “poor quality” DNA.  Some customers have noted a problem with their PCR reactions which are the result of poor quality DNA.  This could be DNA which has been stored for a long time and may have suffered some degradation.  This could be DNA whose source is a WGA reaction (which will sometimes be poor quality if the starting material was not of sufficient quality or quantity).  In these cases the EXTEND reaction results can be sometimes improved by addition of more than the recommended amounts of template DNA.  Quantities of as high as 40 ng of genomic DNA have been implemented by iPLEX in some cases with improved results from lower quantities.

How does one validate assays?

• After one designs the assays, they should be validated.  Validation can help remove/fix problem assays before putting them into production.  You can validate assays by running it in at least 24 wells of a plate.  Two or more wells can be “No Taq controls” to show any primer-primer interactions.  Two or more wells can be “No template controls” to show any DNA contamination problems and the remaining wells can be run with known good DNA (such as commercially available DNA samples).  A performing assay should show good extension yield and clear clusters.  If the assay looks good here, then you can proceed to production with these assays.