Fragment Analysis FAQs

Q1) How do I get the Fragment analysis results and how do I view them?
Ans: The results will be sent through email via a link to our server to prevent email storage deficiency. You can download the compressed file in .zip format (for Windows). The run files were saved as *.fsa files, and the sizing tables are available in *.xls and *.txt files. You may open and analyze the *.fsa files with ABI freeware, Peak ScannerTM Software v1.0 (http://www.appliedbiosystems.com/peakscanner).
If you have requested the report through service SS3001, you would receive the run report(s), which contain the analyzed electropherograms by GeneMapper® v4.0. They are presented in *.pdf files.

 

Q2) What is the accuracy of sizing in Fragment Analysis and how is it different from my manual gels? 
Ans: The accuracy of sizing provided in our analysis is +/- 0.5 bp. We can achieve such high levels of accuracy and automation in sizing because we include size standard in every sample, unlike manual gels where size standard is loaded in only one lane of the gel.

 

Q3) I have fragments that are not in the sizes I was expecting. What can I do?
Ans: Check the sizing precision - display your size standard peaks (up to sixteen different samples) in one display panel, and align by size. All the peaks should overlap exactly. If not, redefine the size standards for those lanes that do not line up, and then recheck sizing precision. Once the size standard peaks superimpose exactly, then the sizing precision is correct and the data can be considered reliable. At 1st BASE, we will perform this check before releasing the raw data to our customers.

 

Q4) What are the specifications for fragment sizing?
Ans: Fragment sizing run times are 45 minutes with single base resolution up to 400 base pairs and 0.15 base pair standard deviation.

 

Q5) What is the size range that can be detected by your fragment analysis service?
Ans: The promised size resolution is the range of: >=75bp and <=490 bp. 
Please write to us separately if you have sample size > 500bp to be analysed using our Fragment Analysis service.

 

Q6) Which size standard should I use?
Ans: Don't worry; we will select it for you.
500-ROX (for 3-dye multiplexing); or 500-LIZ (for 4-dye multiplexing) fluorophores.

 

Q7) Do you have any control for Fragment Analysis?
Ans: Yes, each order will be attached with our control reaction using our control DNA and control labeled oligos, which is Blue (5' 6-FAM).

 

Q8) What dyes can I label my primer with?
Ans: Only 1 primer of your PCR (either Forward or Reverse) need to be labeled: 6-FAM, HEX, TAMRA, or ROX. Please enquire if your dye combination is different from below:

Dye Set D

Dye Colour
FAM BLUE
VIC/HEX/JOE GREEN
NED/TAMRA YELLOW
ROX (Size Standard) RED

Dye Set G5

Dye Colour
FAM BLUE
VIC/HEX/JOE GREEN
NED/TAMRA YELLOW
PET RED
ROX RED
LIZ (Size Standard) ORANGE


Q9) Can I have more than one marker labeled with the same dye?
Ans: Yes, as long as the expected allele size ranges do not overlap, or at least >10bp apart.

 

Q10) Do I have to clean up the samples before I submit them?
Ans: No. Purification is rarely needed. Except samples that contain high salts (usually from RE buffers used in TRFLP analysis) that will interfere with the electro-kinetic injection.

 

Q11) Can I multiplex my PCR reaction?
Ans: Yes. For targets with a similar allele size range, use different dyes (but from the same filter set).

 

Q12) I have multiple PCR products that I want to run in a single injection, should I pool the products before submitting it to the facility?
Ans:

  1. New project: send in separate tube, we will pool for you.
  2. Repeat projects: before customer pool all the different dyes together, ensure similar intensity of each dye and send in 1 plate.

Note: 6-FAM, VIC and TET generate higher signals than NED or HEX. 
For (i) and (ii), the extent of the spectral optimization is different but charges are the same as per dye.

 

Q13) I have tried to run some of my samples at different Dye Set (which is using Filter F) through Applied Biosystems Genetic Analyzer, but I am getting unsatisfactory results. How can that be?
Ans: Applied Biosystems (ABI) has changed the matrix condition bounds of the spectral calibration kit for Filter F (catalogue number: 4345831). Please refer to: 
http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_042198.pdf

Thus, if you are using any amplification kits containing the labeled primers that needs to be run using Filter F, the kit has to be from ABI's brand. If not, you will have to get the matrix calibration kit from your supplier to calibrate your machine separately.

 

Q14) What is the difference between 1st BASE Fragment Analysis Service and those from other service providers?
Ans: We provide full convenience to our customers to optimize the loading prior to actual analysis. We also have our internal PCR control standards using our in-house synthesized labeled oligos for each batch of analysis to ensure the size call consistency.

 

Q15) I noticed some of the size standard is not consistent. How can that be?
Ans: According the user manual of GeneMapper® Software Version 4.0 for Microsatellite Analysis Getting Started Guide (page no. 24): depending on the type of instrument, type of polymer and its primer, it may be appropriate to choose one of the other size standards that omit peaks. Specifically, the 35bp-peak can be eclipsed by the neighbouring primer peak, or the 250-bp and 340-bp peaks can migrate abnormally on the capillary electrophoresis instrument.

For the fragment analysis done by 1st BASE, we set the size range from 50-bp to 500-bp and we omit the 35-bp and 250-bp peaks during the sizing analysis. As such, you would still able to view the 250-bp peak from the raw data without the size call. If you check the sizing precision according to what we described in Question no. 3 of this FAQ session, you would see the abnormal migration for this 250-bp peak. For the 340-bp peak, we found it was still consistent along our runs and thus we did not omit this peak during our size call program.

 

Q16) Would you disclose my project to other parties?
Ans: We understand that some of the fragment analysis projects are confidential. We guarantee the confidentiality of all our services from Fragment Analysis to DNA sequencing services and the purchasing of primers from us. Non-disclosure agreement is also available upon request.