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Services

DNA sequencing

This service is offered to units of the Pasteur Institute involved in public health activities, and to the French National Reference Centers  in a context of bio-emergency (major outbreak, emerging agent, ...).

Contact: pf8-seq@pasteur.fr

General information

Sequencing reactions are performed on the platform, using as starting material the PCR products (in the majority of cases), RT-PCR products or plasmids that are sent by requesting laboratories. Where necessary, the platform can be involved more upstream, by performing PCR or RT-PCR on DNA / RNA matrices.

Amplification products are purified by ultrafiltration (Millipore) and their concentrations determined on agarose gel or by spectrophotometry (Nanodrop). Sequence reactions are prepared in 96 wells microplates using Big Dye Terminator chemistry. Sequence reaction products are purified by ethanol precipitation. Sequences are obtained on a capillary sequencer ABIPRISM 3730XL, are briefly checked and then immediately made available to requesting laboratories.

Confidentiality is ensured at all steps. Our sequencing service  accepts varying numbers of samples but is not intended for very high throughput.


Samples shipment

Reception and packaging of samples
Please define the degree of emergency in order to enable us to establish priorities.
 
- Receipt of samples: all working days of the week
 
- Packaging of samples: if possible,  96 wells microplate format or 8 x 0.2 ml tube strips with a minimum volume of 25 µl per sample (amplified products).

Shipment of samples and attached data sheet
Before providing the samples, please send the data sheet (download data sheet) by e-mail at: pf8-seq@pasteur.fr . Samples must be deposited at the platform (BioTop Building, -1 floor) accompanied by an internal order sheet (BCI) signed by the laboratory head.

Quality of samples
The control of the size and concentration of amplified products must have been done beforehand by the requesting laboratory (please provide gel photographs if possible).
Sample amount and concentration:
        - 20 to 60 ng of a PCR product for each sequence reaction
       - 150 to 200 ng of a plasmid for each reaction sequence
This amount of DNA should be contained in a volume ranging between 1 and 7 µl.

Support for commonly used oligonucleotide primers
The platform can renew stocks of oligonucleotide primers that are used on a regular basis. Other oligonucleotide primers should be provided with the samples.

Purification of PCR products
The purification of PCR products is performed on the platform by ultrafiltration on membrane for product size > 150 bp.
For shorter PCR products, please specify the size so that we can adapt the procedure.

Storage of samples
The products of sequencing reactions are conserved 1 day after electrophoresis and can be re-used in case of technical problem with the sequencer.
Purified PCR products are conserved during three months on the platform, so that sequencing reactions can be repeated when needed.

Results

Time to results
The entire process, from receipt of samples to delivery of sequences, takes an average of 48 hours. In case of bio-emergency, the results can be available in less than 24 hours.

Availability of results
The requesting laboratory is informed of the availability of sequencing results, as well as of a quality assessment, by e-mail.
Sequence chromatograms are made available through a password-protected, lab-specific directory.
Sequences can be edited and/or further analyzed by the platform in particular cases.

Data backups
A back-up of the sequence chromatograms is stored by the platform during a period of 6 months.

Prices

Our current price is 2.50 € per sequence reaction, corresponding to 240 € for a 96 wells microplate.

Microsatellites / MLVA-VNTR

The platform offers a fragment analysis service for typing mini-  and micro-satellites, Variable Number of Tandem Repeats (VNTR), or Multilocus VNTR Analysis (MLVA).

Size determination is performed by using an ABI PRISM 3730XL capillary sequencer with a run time of approximately one hour.
The platform also provides users of this service with software GeneMapper v. 4.0  (Applied Biosystems) allowing an easy and user-friendly data analysis.
Binning of sizes into alleles and further data analysis can be performed on the platform in some cases.

Contact : pf8-seq@pasteur.fr