DNA Sequencing in collaboration with Marine Laboratory at Woods Hole

THIS SERVICE WILL BE DISCONTINUED JULY 1, 2014.

THE LAST DAY TO SUBMIT SAMPLES IS JUNE 30, 2014.

Results will be available on Chromatograph server until December 31, 2014.


Sample Shipment
Sample Preparation
Viewing Sequencing Results
Accessing Sequencing Results

Introduction:
The Brown University Center for Genomics and Proteomics works in collaboration with the Josephine Bay Paul Center at the Marine Biological Laboratory at Woods Hole to provide DNA sequencing services to Brown and Brown-affiliated users at a competitive rate of currently $3.85 per sequencing reaction. Sequencing services will be billed to the designated accounts on a monthly basis. This price will be charged to all Brown and eligible hospital-affiliated laboratories.

The sequencing service is overseen by Christoph Schorl (Christoph_Schorl@brown.edu), Assistant Professor of Research in the Genomics Core of the Brown Center for Genomics and Proteomics. Christoph is assisted by Kirsten Robinette. Patricia Werner manages all administrative aspects of the sequencing service.

All labs who wish to use the service should contact Tricia (MCB_DNA@brown.edu). She will set up a profile on the sequencing web page for each principal investigator. When contacting Tricia please provide your contact information, billing address, and account number to which the fees for the service will be charged. Each PI will receive labels preprinted with codes (three letters unique to each lab followed by a string of 6 unique numbers).

A web site (MCB DNA Tracker) has been created that allows users to enter their sequencing requests and for Brown and MBL staff to track the flow through the system and to manage the charging of fees back to the investigators using the unique label. Once a label has been entered into the system it cannot be re-used.

Since the website has restricted access, all users must have a valid Brown user ID and password. These are the same that are needed to access any other restricted Brown area, such as the electronic subscription in the library, etc. All hospital-affiliated faculty and staff who already have Brown user IDs will be able to access the system immediately. Those who do not have a Brown ID but wish to take advantage of the sequencing service should arrange for affiliate accounts with the Brown Card office. Affiliate accounts can be arranged for anyone with a reasonable need for Brown electronic services.


Sample shipment  (back to top)
Samples will be shipped from Brown every Monday (or Tuesday if there is a holiday) and must be entered into the system and delivered to either the BMC or LMM stockrooms no later than 12:00, noon on Monday (or Tuesday if there is a holiday).


Sample Preparation  (back to top)
To set up reactions, combine in a 0.5 ml centrifuge tube: 500-1000 ng plasmid or 50-100 ng PCR product plus 30 pmol of primer. 
The total volume should be made up to 10 ul using distilled water.
Please measure template and primer concentration accurately since it directly affects the quality of the sequencing reactions. All dilutions should be made in distilled water.
Place one of the unique labels onto each tubes containing a sequencing reaction, which allows to track the samples through the system. Please attach the label without any overhangs and ensure that the label is readable and finally that all tubes are tightly closed.

If a lab submits 24 or more samples (total), they need to be submitted in a sealed 96-well qPCR plate. Individual investigators from one lab may have to coordinate sample submissions, so that the least number of plates are submitted from a single lab. If required due to the number of samples, more than one (1) plate may be submitted. The plate can be sealed using strip tube lids or adhesive film. Please place samples only in "uneven" numbered columns (i.e., 1, 3, 5, etc.) and leave "even" numbered columns free. The ID numbers need to be entered into both the MCB DNA Tracker and the Excel Documentation file. The Excel file needs to be submitted to MCB_DNA@brown.edu by noon on Monday (Tuesday, if Monday is a Brown holiday).

Finished sequence data will usually be posted to the user's server folder on Wednesday afternoon or Thursday morning of the same week, and users will be notified by email of the posted data.


Viewing Sequencing Results:  (back to top)
PCs:
There are two software options in order to view your chromatographs. The first is BioEdit: download and install this free BioEdit software, then go to "file"/"open" and then choose under the "files of type" option: "ABI files". Go to the folder with your sequencing results and open the file of interest from within BioEdit. Using this software it is possible to stretch or compress the width of the peaks as well as the height of the signal, as well as copy it into software such as Microsoft Word. Alternatively, the sequencing data can be obtained by opening the "seq" file with Microsoft Word.

The second available software program which we use is the Sequence Scanner Software v1.0 from Applied Biosystems. This software is also freeware and can be downloaded from the Applied Biosystems website.

Macintosh:
Mac users can open the .ab1 DNA sequencing data files with the MacVector software available through Brown's download page. The most versatile software is the most recent update version MacVector 7.2.3. In this version, when you open the data file there's a pull-down menu on the lower right corner of the screen that lets you format the number of rows of data per page to make a compact print-out.


Accessing Sequencing Results on the File Server:  (back to top)

On-Campus Users:
The folders are located at filesg.ad.brown.edu\chromatographs_shared\files. To access them map a drive (PC) or mount a drive (MAC); if you need help, check with your local desktop support staff. When the login box appears, enter "ad\" followed by your Brown username (e.g. if your username is jcarber, enter "ad\jcarber"), then enter your regular password. You should be able to see everyone else's folders, but you will only be able to access your own.

Off-Campus Users:
You will need to have VPN Access to access the folders using this method. Please click here to find out how to get VPN Access

The folders are located at filesg.ad.brown.edu\chromatographs_shared\files. To access them map a drive (PC) or mount a drive (MAC); if you need help, check with your local desktop support staff. When the login box appears, enter "ad\" followed by your Brown username (e.g. if your username is jcarber, enter "ad\jcarber"), then enter your regular password. You should be able to see everyone else's folders, but you will only be able to access your own.

Off- and On-Campus Users:
You can access results via the web by visiting http://webfiles.brown.edu. You do not need to use the VPN for this method.

When the login box appears, enter "ad\" followed by your Brown username (e.g. if your username is jcarber, enter "ad\jcarber"), then enter your regular password. After you have logged in, navigate to Files->Chromatographs_Shared->Files. You should then see your folder. We suggest you right-click it and select "Create Shortcut". This will create a shortcut on your home page that you can use to go directly to your own folder. Please note that if you are a new user, web access will become available to you the day after your folder is created.