Automated Fluorescent DNA Sequencing in the CGC
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.
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 about 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#2. 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. There are 3 different rates charged for these types of runs - 3730 runs are all $48.00, short length sequence reads on the 3130XL are $17.00, and long sequence reads 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 3100 instrument and analysis protocols are used with the ET Terminators. See the protocol archives for details.
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 5.2 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, DNA Workbench from CLCBio, and everyone on campus has access to the 4 or 5 network licenses of Sequencher. Data files produced by Sequence Analysis 5.2 and saved in analyzed form can be opened in Sequencher, MacVector, or DNA 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 well worth a read.
Automated Fluorescent DNA Sequencing - Background
Automated fluorescent DNA sequencing using capillary DNA sequencing machines like the ABI Genetic Analyzers in the CGC is based on the use of a different color fluorescent dye for each of the four DNA bases. Attaching these dyes to the four dideoxynucleotide terminators used in the standard Sanger chain termination DNA sequencing reaction results in all the fragments ending in one of the four fluorescent dye-labeled terminator corresponding to the dideoxy bases. These fragments are then separated on a liquid denaturing gel pumped into each capillary. The fluorescence is detected as the fragments electrophorese through a transparent section of the capillary which runs in front of a CCD camera, while being excited with laser light. The use of four different dyes allows the sequencing reaction to be performed in a single tube and the resulting fragments to be loaded in a single well, resulting in greater sample capacity per “lane” as compared to what is possible with radio-labeled fragments.
Sequenase (T7) polymerase has been a widely used enzyme for DNA sequencing with radioactive nucleotides because of its high processivity and low error rate. However using this type of enzyme for fluorescent DNA sequencing is not optimal because the amount of fluorescent DNA produced is low and thus a large amount of template is required to produce good fluorescent signals. The enzyme currently in general use for automated fluorescent DNA sequencing is a variant of Taq DNA polymerase. The thermostability of Taq polymerase allows the sequencing reactions to be carried out like PCR reactions, and the reactions are called "cycle sequencing" reactions. Cycle sequencing reactions are analogous to PCRs except only a single primer is used and only single stranded products are generated. The advantage of using the thermostable Taq polymerase over the use of Sequenase is that multiple rounds of sequencing can be performed without the need to add fresh enzyme. This allows the use of much less template DNA, making this the method of choice in the majority of circumstances. Modifications (point mutations affecting amino acid residues in or near the active site) to the Taq DNA polymerase have enabled it to incorporate the fluorescent dye-labeled terminators more evenly and efficiently, resulting in very even peak heights over a sequence read. Attempts have been made to develop polymerases which have some of the advantageous properties of Sequenase but are thermostable like Taq. One of these efforts is described in more detail at http://www.atp.nist.gov/eao/sp950-2/chapt3-2.htm Another important innovation was the introduction of dideoxy terminators that use two fluorescent dye labels to take advantage of fluorescence resonance energy transfer (FRET) (i.e. ABI BigDye). Taking advantage of FRET using the dual-labeled terminators has allowed improvement of signal intensity and spectral separation to the point where very small amounts of templates can be used. Fluorescent sequencing methods are now robust enough that sequencing large templates such as BACS, P1s, etc. that used to be impractical have become reasonably easy, and sequencing plasmids and PCR products is trivial.
A typical processed electropherogram generated using ABI BigDye v3.1 cycle sequencing chemistry and run on a 3100 is shown at right. The G bases are displayed in black, the A's in green, the C's in blue and the T's in red.
The picture below is a screen capture from the data collection software. You can see that the cycle sequencing reaction products from 16 wells of a 96-well plate are being electrophoresed simultaneously. The large pane at the bottom of the screen shows the fluorescent DNA bands which have been separated up to this point in each of the 16 capillaries lined up next to each other. Since the 3100 is a 16 capillary machine, it takes 6 runs to process a full plate of samples.