Sanger Sequencing Custom


Overview

Clients may submit custom templates (difficult templates, BACs, giant fragments, high GC, etc). to be sequenced using a non-standard reaction cocktail. In order to use the custom service, clients must have determined which custom sequencing reaction conditions to select (for guidance, please contact Patrick Warner at umgcseq@umn.edu). The six options listed within the online ordering system are:

  1. DMSO (added by UMGC)
  2. 1 M betaine, 40 cycles, template denaturation
  3. 1 M betaine plus dGTP, 40 cycles, template denaturation
  4. 40 cycles, template denaturation  (HAIRPIN Protocol)
  5. BAC / gDNA
  6. Fosmid / Cosmid
Pricing

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Standard

Service Scale Unit UMN External
Custom Option Sequencing.        
12 ul of template and primer are combined and submitted in 0.5 ml tubes. 1-any sample $10.49 $12.07
Guidelines

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Submission

How to Order


University researchers can place an online order by entering the ONLINE ORDERING SYSTEM. After completing the sample submission form, print and submit with your samples. Samples can be dropped-off at one of our laboratory locations or mailed/shipped.

Forms

Sample Submission Form - UMN researchers

Sample Submission Form - External

Drop-off locations


1-202 Nils Hasselmo Hall (Minneapolis campus)
1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)
20 Snyder Hall (St. Paul campus)

If mailing samples through the United States Postal Service

Please secure and protect samples with padding and address to:

University of Minnesota Genomics Center
1475 Gortner Ave.
123 Snyder Hall
St. Paul, MN 55108
612-625-7736

For shipping using carrier, please address shipment to:

University of Minnesota Genomics Center

1475 Gortner Ave.
28 Snyder Hall
St. Paul, MN 55108
612-625-7736

Deliverables

Results


You will receive an email after results from your entire order have been posted. If you have not received an email within 72-hours of sample submission for Sanger Classic, Tube or Plate Submission or Clean and Load Only, please contact umgcseq@umn.edu and include your order number. If any sample needs to be reprocessed, an email will not be sent until the entire order is complete. Additional processing time is needed for Sanger Quality Check and Sequencing & Primer Addition Sequencing, and Custom Option Sequencing, services.

Results are in two different formats: text (.seq files) and trace (.ab1 files).

For University researchers, data files are uploaded to the order within the online ordering system; "My Account" tab and then the "Result Files" section on the right next to “History".

For researchers outside the University, data files are emailed to the email address provided on the DNA Sequencing Request Form.

Hard copy


For tube submission, hard copies are included.

For plate submission, hard copies are not included. Printing charge is $1.00/page.

Quality Scores


Quality Values: Calculation and display of quality values (QVs) for pure and mixed bases. The QV is a per-base estimate of the basecaller accuracy.

Sample Score: Calculation and display of a sample score is generated from QVs. It is the average quality value of the bases in the clear range sequence for that sample.

Signal/Noise Value: Calculation and display of the value that represents the average signal/noise value of all the bases in a sample.

FAQ

Where can ABI FAST plates be purchased?


Plates can be purchased through the facility by submitting an online order for DNA Sequencing Request (Sanger HTP). Please call the lab at 612-625-7736 when completing the online form. The price will be changed in the online ordering system to reflect the current plate price. A pack of 10 plates can be purchased through ThermoFisher Scientific (formerly named Life Technologies), part number 4346907.

What is the minimum number of samples that can be submitted in a plate?

The minimum number of samples needed for plate submission is 48. If partial plate is submitted, samples need to be loaded from A1-H1, A2-H2, A3-H3, A4-H4, A5-H5, A6-H6, etc. Empty wells must be guaranteed to be clean and empty. Please note this in the additional comments on your DNA sequencing request form. In addition, you must leave two wells open for our internal control (random well noted on plate and request form).

Do you have primer design tips?

Primers used for sequencing are best between 18-22 bases in length with a predicted Tm between 50-55ºC.  G-C content of 40-60% is desirable.  Avoid primers with strong self-dimers or hairpins. Also, avoid primers which are >90% homologous to other regions within your sample.  A BLAST search will alert you to this problem.

The best sequencing data is found 80-150 bases away from the primer. It is difficult to get good basecalling less than 50 bp away from the primer.

Only sequence in one direction.  Submit forward and reverse primers in separate tubes/wells.

Primers may be ordered through our IDT Portal for University researchers. For information, go to Integrated DNA Technologies’ website.

It is important to dilute primers properly. This calculator can be used for primer dilution.

Is there access to primer design software?

Yes, Primer Design software is available through the Minnesota Supercomupting Institute (MSI): Primer Design.

How to clean-up samples?

PCR samples we recommend Qiagen PCR purification kit or Microcon filters.

Plasmid samples we recommend Milipore Qiagen mini-prep columns or Qiagen QIAprep Spin Miniprep kit.

BAC and cosmid we recommend Qiagen products.

Genomic DNA we recommend Promega Wizard Genomic kit.

What is the turnaround time?

We have a 72-hour turnaround time for Classic Sequencing and Clean and Load Only & Electrophoresis services. Additional processing time is needed for Sanger Quality Check and Sequencing & Primer Addition Sequencing, and Custom Option Sequencing, services. If you have not received an email message before the 72-hour turnaround time for Classic Sequencing or Clean and Load Only & Electrophoresis services, please contact umgcseq@umn.edu and include your order number.

What software can be used to view .ab1 files?

The .seq files will open in any text or word program. 

If you are using a Mac and are sent PC formatted .ab1 files, download the following program to convert your PC files to Mac formatted .ab1 files.  PC to Mac Conversion Program. Programs are needed to view .ab1 files. 

  • Sequence Scanner (Windows 2000, XP)
  • Chromas (Windows 3.X, 95, 98, NT, XP)
  • MacVector (Mac OSX)
  • TraceViewer (Windows 98 or newer; Mac 10.1.4 or newer)
  • TraceViewer Archives (Classic Mac OS and UNIX/Linux)
  • EditView (Mac OS 9 or earlier)
  • FinchTV (Mac OS 10.2.8 and above; Windows 98, NT, 2000, XP)

If using phred/phrap, update the phredpar.dat file by adding the following line "KB_3130_POP7_BDTv.3mob" terminator big-dye ABI_3100.  This represents "primer", chemistry, dye type, and machine type.