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DNA Sequencing

Capillary Sanger DNA Sequencing in the CGC

For information on Next Generation Sequencing data analysis capabilities see the Data Analysis page, linked at left

The CGC has two ABI Genetic Analyzers for DNA sequencing, a 16 capillary 3130XL and a 48/96 capillary 3730.   The 3130XL generally runs with an 80cm capillary array and is used primarily for long sequencing reads.  It can also be used for fragment analysis with 1Kb size ladders.  The 3730 runs a 50 cm 48 capillary array and is used routinely for both sequencing and fragment analysis.

Supplies/Costs

The 3130xl Genetic Analyzer is a 16 capillary machine, so each run processes 16 samples as two columns of 8 in a 96 well plate. Before each run fresh polymer is pumped into all 16 capillaries regardless of the actual number of samples being analyzed. Therefore, it is wise to plan runs in groups of 16 samples for best cost efficiency. The data collection software allows users to define analysis methods for each run (set of 16 samples loaded in a plate). Different protocols may be used for different runs in the same plate. This makes sharing plates with other users simple.

36cm capillary array in a 3100

Users are expected to provide their own consumable supplies, such as their BigDye reaction mix and primers, dilution buffer, 96-well plates, and sealing film.  Use of the instrument will always be charged on a per-run basis because that is the basis on which costs are actually incurred. The ABI sign out sheets include the instruction that users sharing a run can divide the charge to different budget numbers in sixteenth of a run increments, but that any empty wells in a run will be divided equally between the users of the run.

The 3730 Genetic Analyzer is a 48 capillary instrument, with one run being half a 96 well plate. Before each run all 48 capillaries are filled with the POP7 polymer this instrument uses exclusively. The capillaries are 50cm long and are spaced apart so that they match up to every other column of the plate. This means a single run uses either all of the odd numbered or all of the even numbered columns of the plate.  A 3730 run currently costs $48.00 so, a dollar per well. For sequencing there are two instrument protocols - fastseq50cm which provides about 6-700 base reads in an hour, and standardseq50 which provides up to 900 base reads in 2 hours. There is also a fragment analysis protocol that works with the 400 and 500 base size standards and runs in an hour. Both dye set D and G5 are available.

The standard configuration of the two ABI Genetic Analyzers currently (2010-11) is a 50cm array with POP7 on 3730 and an 80cm array with POP7 on 3130xl. The 3730 can do fragment analysis and short read length sequences, up to about 600 bp, in about 1 hour per run. The 3130XL does short reads of about 700 bp in 2hours, or long reads of up to 1000 bp in 3 hours. UPDATE - ABI Genetic Analyzer rates as of 1/1/2016: 3730 runs are all $53.00, and 3130XL runs are $22.00.

What you can buy from the CGC:

- BigDye3.1 Ready Reaction Mix from ABI.
- ABI 5x BigDye reaction buffer.
- Flat Top flexible 96-well plates.  These can be used for PCR reactions, cycle sequence reactions, and for loading samples into the 3100, 3130xl or 3730.
- Plate sealing film, for running PCR and cycle sequencing reactions.
- ABI plate sealing septa, for loading plates in the 3130xl or 3730.
See the CGC Store list for the details.
- DyenamicET Terminator reaction mix from Amersham (now GE Healthcare) can also be used on the ABI sequencers in the CGC. The reaction mix has to be purchased directly from GE Healthcare. A different PCR cycle program and different 3130XL instrument and analysis protocols are used with the ET Terminators. See the protocol archives for details.

Practical considerations
The ABI Genetic Analyzers come with a software package which allows the computer to operate the machine and collect raw data files from it, the DataCollection 3.0 package. For sequence analysis you will be using Sequence Analysis 5.3 to interpret the raw data and call the bases. For assembling and aligning sequences the Center has MacVector with Assembler (v.10), SeqScape from ABI, Main Workbench from CLCBio, and everyone on campus has access to the 4 or 5 network licenses of Sequencher. Data files produced by Sequence Analysis 5.3 and saved in analyzed form can be opened in Sequencher, MacVector, or Main Workbench; The old Edit View software from Perkin Elmer does not work with data files produced by DataCollection 2.0/3.0 or Sequence Analysis 5.2.
It is also worth checking the DNA sequencing section of the protocols archive page to see if another user may have found a better or less expensive way to do the kind of experiment you have in mind. The center posts useful protocols and user tips on that page when a user finds a method or experimental design they think is especially good.  For a pretty comprehensive coverage of how DNA template quality affects sequencing results, and how to  improve results, have a look at the QIAGEN Guide to Template Purification and DNA Sequencing It is long but worth a read.

 

 

 

 


Date last changed Jan 31st, 2017 @ 14:06:22 PST