The ABI platform is the "Gold Standard" for producing high quality sequence data for many traditional types of projects. The machines provide reads of up to 800 high quality bases, and are extremely sensitive and reliable. They support high throughput resequencing of particular regions of interest in any genome, though most of our work involves either human, mouse, or bacterial genomes. They can produce approximately 1500 reads per day, per machine (1.2 Megabases of sequence per machine).

Services

• Automated primer design
• PCR amplification
• Sequencing reactions
• Sequencing detection, ABI 3730xl
• Data assembly
• Sequence variation identification and mutation detection
• Fragment analysis
• Fee for service sequencing

The Sequencing Group sequences human, mouse, and bacterial DNA. For PCR to detection projects, we perform two rounds of sequencing to ensure the highest pass rate. A first round of sequencing is performed across all samples and amplicons using carefully designed primers specific to the region of interest. If the pass rate for the first round of sequencing is less than 90% (for human and bacteria; 85% for mouse) a second round of sequencing and troubleshooting, utilizing different chemistries and sometimes redesigned primers, is guaranteed. Often we have good data for amplicons which yielded poor quality data after the first round. We frequently achieve higher that 90% final success rate on our sequencing endeavors.

Sample Submission Requirements

Sample Requirements:

1. Minimum of 25 ng per amplicon of DNA is required for each sample.
   
   
2. Samples submitted in plates may be at different volumes. However, all samples MUST be normalized to one standard concentration (e.g. 25 ng).
   
   
3. All tubes must be clearly labeled with sample names as entered on this Sample Submission Form (use attachments for more entries). Print the form for submission.
   
   
4. If sequencing more than 5 samples, samples MUST be submitted in 96 well plate (Thermo-Fast 96 Detection Plate, Abgene Cat# AB-1100). Please leave wells A1 and H12 empty for controls. Plates should be sealed secure (Abgene AB-0558 Adhesive PCR Film).
   
   
5. Pack plates (samples) in dry ice in a styrofoam (or other insulated) cooler. Make sure there is dry ice on the top, bottom, and both sides of the plates. If shipping more than 1 plate, wrap plates in aluminum foil and/or vinyl tape so they will not move around during shipment.
   
   
6. Sample Submission Form MUST be included with all shipments. Sample names must not contain anything besides numbers and letters (i.e., no underscores, dots, dashes, spaces, etc.)
   
   
7. Upload the Sample Submission Form to GIGPAD. Once you receive an internal project name from Dr. Iartchouk, click on "Study Name" link from the "Study Listing" page in GIGPAD. On the "Study Details" page, click on the "Data Files" link. Upload the Sample Submission form using the "Upload Files" link.
   
   
8. Please mail samples & form to:
   Soumya Vemuri
   Partners Center for Personalized Genetic Medicine
   65 Landsdowne Street, Suite 350
   Cambridge, MA 02139
   
   

Note: Please visit https://portal.hpcgg.org/ to request access for our Gateway for Integrated Genomics and Proteomics Applications and Data (GIGPAD) database.


Fee for Sequencing Submission Requirements:

1. Products should be completely ready to run on the ABI 3730xl with no further processing from the Sequencing core.
   


2. Products MUST be in 384 well plates (Thermo Scientific Thermo-Fast Diamond 384, Cat # AB-1111).
   


3. Plates MUST be submitted with the Fee for Sequencing/Fragment Analysis Sample Submission Form. Form should be uploaded to GIGPAD (see instructions above).

Fragment Ananlysis Submission Requirements:

1. PCR products should be sent ready for detection after addition of Liz 500 Size Standard by the sequencing core.
   


2. Plates MUST be submitted with the Fee for Sequencing/Fragment Analysis Sample Submission Form. Form should be uploaded to GIGPAD (see instructions above).

Plasmid Sequencing Submission Requirements

1. Plasmids must be normalized to 20ng/ul
   
   
2. Our sequencing facility has the following primers available for plasmid sequencing:
   T7 Terminator
   T7 Promoter
   SP6
   M13F
   M13R
   T3
   All other plasmid specific primers must be sent along with plasmids; all primers must be normalized to 10uM
   
   
3. Plates MUST be submitted with the Plasmid Sequencing Sample Submission Form. Form should be uploaded to GIGPAD (see instructions above).
   
   

PCR Product Submission Requirements:

1. PCR products should be sent ready for BIG DYE terminator cycle sequencing -
   OR
   If cleanup is needed make sure this is clearly noted in the sample submission form
   
   
2. A gel must be run to prove success of PCR reaction and presence of product in samples; a copy of this gel picture MUST be sent with samples.
   
   
3. Plates MUST be submitted with the PCR Product Sample Submission Form. Form should be uploaded to GIGPAD (see instructions above).
   
   

For project design and price quotes please contact:
Oleg Iartchouk (Ph.D)
Partners HealthCare Center for Personalized Genetic Medicine
65 Landsdowne St, Room 350
Cambridge, MA 02139
Email: oiartchouk@partners.org
Phone: 617-768-8377
Fax: 617-768-8510