SANGER Sequencing

Instructions for Submitting Samples for SANGER Sequencing

Fees:
$3.90 per sample template*
$11.76 per sample template for External Academic Users.

*Rate charged to URI, RI-INBRE & RI-EPSCoR users for FY2017. Financial assistance has been provided by RI-EPSCoR (NSF Award EPS-1004057) and the College of the Environment and Life Sciences at URI. Rates effective: 7/1/2016

URI Users:

Use the GSC as the vendor (PeopleSoft Vendor ID = URI_GSC).

For a one-time submission or use, a user’s department must provide an Internal Vendor (IV) Purchase Order as payment for services or equipment use.  Please remember to have the department forward the IVPO to the GSC.

For multiple submissions or use, a user must issue a College Requisition for a Blanket Purchase Order as payment and state “Blanket Requirements: start date – end date” in the description to cover the number of samples or hours of service you need during the time period.  URI Purchasing will then issue the Blanket Purchase Order to the GSC.

Additional information for internal vendor payments is available from the Office of the Controller.

External Users:

Please provide a PURCHASE ORDER with the RI Genomics and Sequencing Center as vendor as payment for services. Please also include your billing address.

Sample Preparation:

Follow our Sample Preparation Guidelines in order to optimize your template and primer for the Applied Biosystems BigDye® Terminator v3.1 chemistry. Template purification and quantification are the most critical factors in obtaining good sequence data. If you are unsure of the quality of your DNA, please check on an agarose gel before submitting the sample. We are not responsible for poor sequences due to impure templates.

Sample Submission:
Fill out a Submission Form to identify your samples and template type. To facilitate billing and file distribution, please provide all the information at the bottom of the form: Name, Principle Investigator, Department / Institution, PO Number and email address.

Submission Form generator is also available that will calculate the amounts of template needed for both PCR products and plasmid templates. The instructions are very easy to follow and will result in a submission form that can be printed from your web browser.

Target amounts for dsDNA templates:

  • PCR products: 2.5 ng DNA per 100 bases per reaction
  • Plasmids: 300-500 ng DNA per reaction

Primer amount:

  • Use one primer only; either forward or reverse, but not both!
  • 5 pmol per reaction (Note: 5 pmol = 2.0 µl of a 2.5 µM stock)

Single sample volume:

  • 12 µl per reaction; add template plus one primer in the amounts above to MB grade water.

To facilitate pipetting, submit your sample in duplicate with a total volume of 24 µl.

Submit your template and primer combined in a 0.5 or 1.5ml tube. DO NOT submit samples in individual 0.2 ml (200µl) tubes. When submitting 16 or more samples, please submit them in 8-tube strip-tube(s) (capped) or a 96-well plate (capped or sealed).

If submitting cosmid, BAC, or genomic DNA templates, please call for more specific information regarding amounts.

Labeling Samples:
Please identify your samples using the following code (6 character limit): Your initials followed by the number 1 to 9999 (i.e.: PJ1, PJ2, etc.). You should increment the number with each submission. This code facilitates instrument plate setup and data file management.

Sample Analysis:

Sequencing on the AB 3130xl or 3500xl genetic analyzers is routinely conducted using POP7 polymer, a 50 cm long capillary array and the KB Basecaller software. These conditions normally produce high quality sequence that extends to 800-1,000 bases.

PCR products less than 900 bp in length will be analyzed using an analytical protocol that looks for the end of the raw data. Please identify your PCR product and its size on the Submission Form so this protocol may be specified during instrument setup.

ALL sequence data will be examined for quality and content. Please note that sequence data are NOT trimmed or edited!

Results:

Sequence data will be exported in the standard Applied Biosystems formats, ab1 and seq (ABI and FASTA text only sequence), and distributed by email upon completion of the run. Formats scf and phd.1 (Phred) are also available at the request of the user. Please provide an email address with sufficient capacity to receive your data.

Examining Your Results:

We encourage users to critically evaluate their raw data for both successful and failed sequences.  One way to view your sequence data is with the free Applied Biosystems Sequencer Scanner software (Windows PCs only).  This trace editor and viewer is a very useful tool that enables examination of raw data as well as the text sequence and electropherograms.

FinchTV by Geospiza is a free trace viewer for DNA sequences in ab1 and scf file formats on Linux, MacOS X, Windows, and Solaris platforms.  This chromatogram viewer can display an entire trace in a scalable multi-pane view and also has raw data views, BLAST searching and the ability to reverse complement sequences and traces.

Two other free programs for examining your sequences are 4Peaks and Chromas Lite.

Schedule:

The schedule for sequencing is posted on the GSC Calendar. Please deliver your samples to 352 CBLS building by 8:30 AM. If you have any questions or problems, call the RIGSC manager at 874-5919.


Sample Preparation Guidelines For SANGER Sequencing

In order to optimize your template and primer for the Applied Biosystems BigDye® Terminator v3.1 chemistry, please consider the following:


DNA Template:

Template preparation is the most critical factor in obtaining good sequencing data. If you are unsure of the quality of your DNA, please check it on an agarose gel or with the NanoDrop before submitting the sample.

Purification – QIAGEN’s QIAwell and QIAprep kits are recommended for purifying plasmids and the QIAquick kit for PCR cleanup. The template should have an OD260/OD280 ratio close to 1.8. Make sure there is NO RNA in the sample.

Quantification – The quantity of template to be used in the sequencing reaction is given below. Quantification can be achieved by spectrophotometric determination (see Procedures) or by commercial assay kits such as Quant-it by Molecular Probes. Alternatively, we suggest you consider using the Nanodrop 8000 instrument in 352 CBLS. The Nanodrop spectrophotometer uses only 1 µl of sample in determining both the concentration and purity of DNA and can also measure up to eight samples simultaneously.

Resuspension & Dilution – Use sterile deionized water (molecular biology grade) or 10 mM Tris-HCL (pH 8.5). EDTA should NOT be present as it can inhibit the sequencing reaction.

Target amounts for for dsDNA templates: 

  • 2.5 ng DNA / 100 bases per reaction for PCR products
  • 300-500 ng DNA per reaction for plasmids


Primer:

The sequencing primer should be used at a concentration of 5.0 pmol per reaction.

Primers can be ordered from a number of different vendors. Please follow the suggestions below in order to optimize your primer.

Selecting Sequencing Primers – The choice of sequencing primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions with this kit. These decisions are particularly important when sequencing is done on real-time detection systems where signal strength is critical. Some of the recommendations given here are based on information that is general knowledge, while others are based on practical experience gained by Applied Biosystems scientists.

Optimizing Primer Selection – The following recommendations are provided to help optimize primer selection:

  • Primers should be at least 18 bases long to ensure good hybridization.
  • Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided.
  • Keep the G-C content in the range 30-80%.
  • For cycle sequencing, primers with melting temperatures (Tm) above 45 °C produce better results than primers with lower Tm.
  • For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm >45 °C.
  • Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
  • Avoid primers that have secondary structure or that can hybridize to form dimers.
  • Several computer programs for primer selection are available. They can be useful in identifying potential secondary structure problems and determining if a secondary hybridization site exists on the target DNA. We suggest that you consider using Lasergene’s Primer Select for this task (see Lasergene under the Services menu)

The BigDye® Terminator v3.1 Cycle Sequencing Kit – Protocol (©2002 Applied Biosystems) also contains the following information which may help in your sample preparation:

DNA Quality:

Poor Template Quality
Poor template quality is the most common cause of sequencing problems. The following are characteristics of poor quality templates:

  • Noisy data or peaks under peaks
  • No usable sequence data
  • Weak signal

Always follow recommended procedures to prepare templates.

Contamination
Potential contaminants include:

  • Proteins
  • RNA
  • Chromosomal DNA
  • Excess PCR primers, dNTPs, enzyme, and buffer components (from a PCR amplification used to generate the sequencing template)
  • Residual salts
  • Residual organic chemicals such as phenol, chloroform, and ethanol
  • Residual detergents

Determining DNA Quality
The following methods can be used to examine DNA quality:

  • Agarose gel electrophoresis. Purified DNA should run as a single band on an agarose gel.
    Note: Uncut plasmid DNA can run as three bands: supercoiled, nicked, and linear.
  • Spectrophotometry. The A260/A280 ratio should be 1.7 to 1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. Agarose gels reveal the presence of contaminating DNAs and RNAs, but not proteins. Spectrophotometry can indicate the presence of protein contamination, but not DNA and RNA contamination.
  • Agilent 2100 Bioanalyzer: Agilent’s Lab-on-a-Chip technology utilizes a network of channels and wells that are etched onto a glass or polymer chip for sizing, quantification and quality control of DNA, RNA, and proteins. Only 1 µl of sample is used in each analysis with 12 samples analyzed per chip. Results are delivered within 30-40 minutes in high quality digital data and can be shown in gel-like image, electropherogram and tabular formats.