Massachusetts General Hospital Sequencing Core MGH DNA Core Facility Center for Computational and Integrative Biology Directions to the Sequencing Core Sequencing Core - DNA CORE - Molecular Biology - Massachussetts General Hospital
 
 

 

Welcome to the MGH DNA Sequencing Core

Stephen Belmonte, Manager
38 Sidney Street, Suite 100
Cambridge, MA 02139
phone: (617) 726-5982
fax: (617) 726-0077

If you are visiting our website for the first time, you will find information about sample submission and data retrieval for the sequencing core by clicking on the appropriate link under "Sequencing Core Overview."

To create a new a new user account or to login to the system, please click on the link below. If your results are available, you will see a link "Sequencing Results" at the top right part of the page.

Login / New User Registration



Methods

DNA samples are sequenced with Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kits which use a fluorescently-labeled dideoxy-nucleotide chain termination method. After cycle sequencing and clean up, the DNA samples are then resolved by capillary electrophoresis on an ABI 3730XL DNA Analyzer which translates the fluorescent signals into their corresponding base pair sequence.

Range & Accuracy

High quality DNA templates generally yield sequence read lengths of up to 600 base pairs, with a cumulative error rate generally less than 1% over the first 400bases. Range and accuracy are critically dependent on the purity and quality of the DNA.

Sample Preparation

DNA Templates

As previously noted, purity and quality of your DNA template play critical roles in obtaining good sequence data. It is absolutely essential that all templates are free of salts and other contaminants which can greatly affect the quality of the sequencing results. Most commercial DNA preparation methods have been known to give reliable data when the protocols were carefully followed.

Best results are obtained with DNA samples having a 260/280 OD ratio of 1.8 or greater.

DNA samples must be diluted in dH2O to the following concentrations:
Double-stranded plasmids: 0.2 µg/µL
Cosmids or lambda clones: 0.4 µg/µL
Single-stranded phage: 0.1 µg/µL
PCR products: (less than 400bp long) 20 ng/µL per 100 base pairs of product
PCR products: (over 400bp long) Calculate your concentrations as you would for a plasmid.

Primers

Universal or custom sequencing primers must also be submitted mixed in the same tube or well with template DNA samples. For best results, primers should be designed with the following in mind:
  • Primers should be submitted at a concentration of 10ng/µL, premixed in the same tube or well as the template
  • Primers should be between 18-24 nucleotides in length
  • Primers should have a calculated Tm of greater than 50°C but less than 65°C
  • Avoid runs of a single base (e.g. 3 to 4 repeats of the same base)
  • Avoid primers with secondary structures or the potential to self-hybridize

Sample Submission

To minimize costs, handling time, turnaround time, and the potential for error, we request that all samples are submitted according to the following guidelines. Samples that require extra handling may be delayed.

  • Primer and template must be premixed in the same tube or well at a volume of at least 20µL.
  • Printed submission form must accompany every order. Barcode should appear near the top left corner.
  • Samples should be submitted in 1.5mL flip-cap tubes or in a 96 well, "V" bottom plate. Please seal plates with covering that is easily removed (no heat-seal membrane or strip caps please).
  • Sample names must be written clearly/legibly/neatly on the tops of the tubes. No stickers please.
  • Sample names cannot be more than five characters; longer names will be truncated. We highly recommend that sample names be kept simple and consecutive.
  • Sample tubes must be racked in the order listed on the submission form. (This is our most frequent cause of "extra handling." When combined with illegible labels, complex naming conventions, and large order, this can definitely cause samples to miss the daily queue.)
  • Samples submitted at drop off sites (see below) should be placed in the supplied Nalgene boxes with the submission sheet secured to the box with a rubber band. If boxes are in short supply, please tape tubes to submission form in the order listed.
  • Do not place tubes loosely in a plastic bag. Your turnaround time may be delayed.
  • If submitting by mail, tubes must be taped to paperwork in the order listed on the submission form. Use additional sheets as necessary.
  • Air mail submissions are best submitted in sturdy plates sealed with strip caps. Tape sealed plates often leak during transport.
  • For special circumstances, please contact us for recommendations.

Premixed Reactions using ddH2O

For Double Stranded Plasmid DNA:
  • 10µL of DNA template at a concentration of 0.2µg/µL
  • 10µL of Primer at a concentration of 10ng/µL
  • Final volume will be 20µL.
For PCR Products (less than 400bp):
  • 10µL of DNA template at a concentration of 20ng/µL per 100 base pairs of product added to:
  • 10µL of Primer at a concentration of 10ng/µL
  • Final volume will be 20µL

High Volume Sequencing

96 Well Format: High Volume Sequencing

If greater then 48 samples are being submitted for sequencing, you must submit in plate format (please use 96 well, "V" bottom plates). If you are submitting an entire 96 well plate for sequencing, enter a plate name and the individual sample names will correspond to their respective positions on the plate. (i.e. A1, A2, A3, etc) If you are submitting less than a full plate, arrange your samples in rows starting in well A1. (Refer to the figure below.) DO NOT LEAVE EMPTY WELLS BETWEEN SAMPLES. Primer and template should be pre-mixed in the same well at a volume of 20µL.

384 Well Format: High Volume Sequencing

Contact the Core if you would like to submit samples in 384-well plate format. The online submission form is not set up to officially accommodate 384-well plates.

Sequencing Core Drop-off Points

Samples are processed in the order they are received. Results are posted 24 to 48 hours after we receive them. Rapid turnaround depends on all users following the submission guidelines outlined above. Samples with properly completed submission forms can be submitted to the Sequencing Core at the following locations:

  • For those who work in the Simches Building: on the 7th floor in the central corridor just outside the doors of the elevator lobby.
  • All others on the main MGH campus: Jackson Building, 13th floor, room 1302.
  • Charlestown campus: Room 8223, CNY 149 8th floor center, south side.
Samples are picked up by a courier service and brought back to us every morning, Monday through Friday. You may also mail your samples to:
    MGH DNA Sequencing Core
    38 Sidney Street
    Suite 100
    Cambridge, MA 02139
For submissions to the Simches Building 7th floor, Jackson 13th floor and the Charlestown Navy Yard please place DNA samples in the Nalgene boxes provided there. In Charlestown the Nalgene boxes must be put inside the cooler in the Sequencing refrigerator or else the samples will not be picked up.

Results

Sequencing results are posted on our website following the completion of each run cycle. They are available in two formats:

  1. Text-only sequence file (distinguished by the .seq at the end of the file name)
  2. Chromatogram/Trace file (distinguished by the .sit at the end of the file name)
Sequence data is left on the website for 3 months, after which it is deleted and cannot be retrieved. Users should download their data to a disk or hard drive to maintain a hard copy of their results for later referral. Chromatogram files must be decompressed before viewing. A program that allows viewing and editing of the chromatogram files is also needed. Several commercial programs, in addition to the freeware programs listed below are available to allow viewing and editing of the chromatogram files. Follow the steps outlined below to set up your computer to take advantage of this feature.
  1. Go to Account creation page to set up an account for data retrieval. You must have an account setup prior to sample submission.
  2. Download and install StuffIt Expander (PC) or StuffIt Expander (Mac) on your computer to decompress your chromatogram files. (Follow you click on the chromatogram file name (*.sit), the file is downloaded from our server and Stuffit Expander saves the file to the desktop or to a location set by you in the Preferences of Stuffit Expander.
  3. Chromatogram Viewers-- 3 freeware programs are listed below. Please install the appropriate viewer on your computer.
  4. Anyone using a Macintosh platform is required to download conversion software in order to view the chromatogram files. The following link will enable you to download the ABI PRISM 3100 Genetic Analyzer Conversion Utilities with instructions: Windows to Mac conversion software.

Turnaround Time

Samples are entered into the queue in the order in which they are received. Samples are collected until noon for that day�s queue. Results are posted 24-48 hours after they are received or as soon as possible. Rapid turnaround depends on all users following the submission guidelines as outlined above.

Consultations

Consultations with facility personnel are possible but, because of time constraints, should be scheduled separately.

Rates

Pricing effective Jan 1, 2009

  • Partners researchers: $2.75 per sample ($1.75/sample for orders containing 96 samples in plate format)*
  • Researchers outside the Partners network: $4.80 per sample ($3.05/sample for orders containing 96 samples in plate format)*
    •
* Please note: These rates apply even if the samples fail to produce useful sequence data unless control samples included with every run also fail. An order containing less than 96 samples will not be charged the lower rate even if submitted in a 96-well plate format.


 
 

  (617) 726-5982