Overview

The HCGS DNA Sequencing Facility is here to facilitate your sequencing needs by providing sequence data in a timely, economical, and reliable manner. The facility is staffed by Joe Anderson and directed by Kelley Thomas and an oversight committee.

We operate a single ABI3130 genetic analyzer on a first-come, first-served basis. This system can run a wide range of sequencing and fragment analysis procedures. Samples can be provided as templates for sequencing or as processed samples (sequences or fragments) ready to run.

DNA samples are sequenced using Applied Biosystems BigDye Terminator Cycle Sequencing Kits (v1.1 and v3.1). After cycle sequencing and clean up, the DNA samples are then resolved by capillary electrophoresis on an ABI 3130 DNA Analyzer which interprets the fluorescent signals into the corresponding DNA sequence.

Links to PDF files for the ABI Chemistry, which include protocols and sample preparation are below:

DNA template quality is extremely important for obtaining good sequence data. The best sequencing results are obtained with DNA samples having a 260/280 OD ratio of 1.7-1.9. Please note on the submission form when you are submitting PCR PRODUCTS (less then 400bp) or if GC CONTENT IS GREATER THAN ~65%.

Below is a table with the recommended amounts of DNA to use in sequencing reactions:

Template	Quantity
PCR product 100-200 bp 200-500 bp 500-1000 bp 1000-2000bp >2000 bp	1-3 ng 3-10 ng 5-20 ng 10-50 ng 20-50 ng
Single-stranded	20-50 ng
Double-stranded (including high copy number plasmids)	150-300 ng
Cosmid, Fosmid, BAC	500-1000 ng (0.5-1.0 μg)
Bacterial genomic DNA	2000-3000 ng (2-3 μg)

Furthermore, primers used for DNA sequencing should be 18-24 bp in length. For sequencing of PCR products, single-stranded DNA, and double-stranded DNA, the recommended working concentration of primer is 2μM; for cosmid, fosmid, and BAC sequencing, the recommended working concentration of primer is 5 μM, and primers should be provided in those concentrations.

When we do sequencing reactions here, per reaction, we use 1μL of sequencing mix, 3μL of sequencing buffer, 2μL of 2 or 5 μM primer, template in the appropriate concentration, and the final volume of the reaction should be 10μL.

All researchers who want to use the services of the Sequencing Facility must register. To register, you should go to http://DNAcore.unh.edu/register.php.

To provide the best, most efficient service, we request that all samples be submitted with primer and template premixed and clearly labeled in a single tube for each sample in a volume no greater than 6 μL. For users submitting samples in a plate format, please use the 96-well format of row/column position (A1-H1, A2-H2,… not A1-A12, B1-B12…), etc. ). An excel template (CLICK HERE) is available. It is important that users follow these guidelines to reduce turn-around time and avoid delays in processing your samples.

Samples are entered into the queue in the order in which they are received. They are then processed as soon as there are enough entries for a run (currently as few as 4 samples). Results are usually returned 24-48 hours after submission. Rapid turnaround depends on all users following the submission guidelines as outlined above.

Samples can be left in the refrigerator on the second floor in Rudman or brought to HCGS on the fourth floor of Gregg Hall.

Once you are logged in if there are results available you will see a link "Sequencing Results" at the top right part of the page. When you click the link you will see a list of all results available and you can save them to your computer. As soon as the website is functional, sequencing data will be placed for retrieval on our website upon analysis in a compressed (*.zip) format when users log in. When this file is uncompressed, the sequence results will include the following files for each sample:

Sequencing data is left on the website for 12 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 use.

You'll also need to download the platform appropriate software for editing and viewing your results: Finch TV (for PC, Mac, Linux), 4Peaks (for OSX), Edit View (for Macs), Chromas and Peak Scanner (for PCs). You will also need a version of StuffIt Expander to uncompress your data once you retieve it from the server.

Due to ABI upgrades to Sequence Analysis software, all those utilizing a Macintosh platform are required to download conversion software 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.

Furthermore, it is now recommended that chromatograms be viewed on a PC platform. Due to the limitations of Macintosh viewing software and the discontinuance of Sequence Analysis software upgrades for Macintosh computers, the PC-based viewers will be better able to display sequence data; however, EditView software for Macs will continue to function, but at a lesser quality for some sequences.

If you want to discuss protocols or results in person, please feel free to contact the core facility personnel to schedule a time to do so. We are happy to discuss your data and samples with you, but may not have time to do so if you just call or drop by (there are many users compared to the staff). Otherwise, we suggest sending an email (hcgs.core@unh.edu).

Sequencing facility rates will be reviewed quarterly by the oversight committee.

UNH HCGS DNA Sequencing Core Facility