Fragment Analysis FAQs

GENERAL

I would like to place order, may I know who should I contact?

You can email or contact us as below:


Singapore

sequencing@axilscientific.com

+65 6775 7318

Malaysia

sequencing@apicalscientific.com

+603-8943-3252

Other Countries

Contact our respective sales representative in your region

Contact our respective sales representative in your region

 

What is the cost for the services?

Please send in your completed order form (you may download here) to our respective email accounts based on your location. A quotation will be followed up after.

 

Can I send orders outside of Singapore and Malaysia? How do I go about doing it?

Fragment order collection points are set up in a few locations in Singapore and Malaysia regions. Please contact our respective sales representative to find out more or if you wish to set up a new collection point.

Singapore

Sequencing order collection point available in most institute. Free local collection.

Malaysia

Kuala Lumpur/ Klang Valley: Our dispatch will collect at your location
Outside of Kuala Lumpur and Klang Valley: Please ship to us via local courier service.

E.g Skynet / CityLink / Poslaju etc.

Others Countries

Please contact the local distributor in your respective countries

 

What is the difference between 1st BASE Fragment Analysis Service and those from other service providers?

All results and reports will be kept confidential. Non-disclosure agreement is available upon request.

 

SPECIFICATION AND RESULTS

What is the Fragment Analysis workflow?

Sample received in our facility > Send Order Acknowledgment > Optimization > Examination with GeneMapper > Run in DNA Analyzer > Analysis with GeneMapper > Run Report > Result Release 

 

What are the specifications for fragment sizing?

Fragment sizing run times are 45 minutes with single base resolution up to 400 base pairs and 0.15 base pair standard deviation.

 

What is the size range that can be detected by your fragment analysis service?

The promised size resolution is between the range of ≥75bp and ≤490 bp.
Please contact us separately if you have sample size > 500bp to be analysed using our Fragment Analysis service.

 

Which size standard should I use?

You can indicate the size standard to be used in the order form. Alternatively, we will select the size standard based on your experiment setup. We provide the following size standard:

500-ROX (recommended for 3-dye multiplexing)
500-LIZ (recommended for 4-dye multiplexing)

Please enquire for other size standards.

 

I have fragments that are not in the sizes I was expecting. What can I do?

Please check the sizing precision first. After which, 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 it doesn’t, please redefine the size standards for those lanes that do not line up, and then re-check the 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.
 

Do you have any control for Fragment Analysis?

Yes, each order will be attached with our control reaction using our control DNA and control labelled oligo, which is Blue (5' 6-FAM).

 

 

What dyes can I label my primer with?

Only ONE primer of your PCR (either Forward or Reverse) need to be labelled:
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

 

Can I have more than one marker labelled with the same dye?

Yes, you can. However, do ensure that the expected allele size ranges do not overlap, and be at least >10bp apart.

 

Do I have to clean up the samples before I submit them?

No, you do not need to purify your PCR product. Purification is only required when your samples contain high salts (usually from RE buffers used in TRFLP analysis) which will interfere with the electro-kinetic injection.

 

Can I multiplex my PCR reaction?

Yes, you can. For targets with a similar allele size range, please use different dyes. However, do ensure it is from the same filter set.

 

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?

  1. For new projects, please send in separate tubes and we will do the pooling for you.
  2. For repeat projects, please ensure similar intensity of each dye before pooling all the different dyes together. Please send in 1 plate.

Note: 6-FAM, VIC and TET generate higher signals than NED or HEX. 
For (1) and (2), the extent of the spectral optimization is different but charges are the same as per dye.


What is the accuracy of sizing in Fragment Analysis and how is it different from my manual gels?

The accuracy of sizing provided in our analysis is +/- 0.5 bp. We can achieve much higher level of accuracy and automation in sizing as size standard is included in every sample, unlike manual gels where size standard is loaded in only one lane of the gel.

 

How do I get the Fragment analysis results and how do I view them?

Results are downloadable via an email with the password protected weblink to our server.
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 will receive the run report(s), which contain the analyzed electropherograms by GeneMapper® v4.0. They are presented in *.pdf files.

 

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. Why is this so?

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, please ensure that you are using the recommended amplification kits from ABI for filter F. Else, you would need to provide us with the matrix calibration from your supplier to calibrate our machine separately.

 

I noticed some of the size standards are inconsistent. Why is this so?

Based on 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 250bp and 340bp peaks can migrate abnormally on the capillary electrophoresis instrument”.

For the fragment analysis done by 1st BASE, we set the size range from 50bp to 500bp and we omit the 35bp and 250bp peaks during the sizing analysis. As such, you would still able to view the 250bp 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 250bp peak. For the 340bp peak, we found that it is still consistent along our runs and thus we did not omit this peak during our size call program.