Shortly after I posted a long-read protocol with 80cm capillary + POP5, another long-read protocol with 50cm capillary and POP7 appeared (Detwiler et al 2004). Recently I tried their protocol and found it is more efficient than 80cm + POP5 with shorter run time and longer read. Some months ago, however, the price of POP7 for ABI3730 was raised 2.5 times in Japan, and it is now more than four times as expensive as POP6 for ABI3700. It is still five times as cheap as original POP6 for ABI3100, though.

Polymer Exchange
When POP7 is first filled into cap array which has been used with POP6, do array fill a few times to replace polymers completely for the safe. Use the array fill wizard on the wizard menu of the data collection software.

Spectral Calibration
The calibration data for BigDye on 50cm capillary + POP6 works fine also with POP7. This data may have been prepared by a technical service people on delivery. If you are concerned about the performance of calibration data on different polymer, you can make it yourself on POP7. An example of spectral calibration for ET kit is as follows. The methods for spectral calibration and mobility installation of ET kit follow the methods for ABI3730/3730XL on an Amersham's web site (63005453.pdf).

Making Calibration Standard
The "Sequence Standard" for ABI3100 is a set of 16 sequence products of M13 or some plasmids with the sequencing chemistry of the dye set to be calibrated. Make 16 sequence samples by 1/20 economy reaction, or four by the standard reaction condition with the ET kit (for BigDye, make 16 samples by 1/40 economy reactions). Ethanol precipitate, dry-up, and store dark and cold. These sequence samples are the "Sequence Standard" of the ET chemistry. To run the standard, add 12uL of formamide to each well (or 48uL and aliquot four times for the standard reaction), vortex, transfer to a plate set onto the machine. If the sequence reaction was made in the ABI reaction plate, it is unnecessary to transfer.

Editing Protocol
(1) Make a new electrophoresis module of an appropriate name (Spect50_POP7_1 for example) with the Module Manager on the data collection software. Edit the electrophoresis condition according to Detwiler et al (2004) with Spect50_POP6 as a template.
  Oven temperature            60^oC
  Voltage of pre-run and run  8.5kV
  Injection time              40sec
  Data delay time            240sec




(2) Make a new instrument protocol of an appropriate name (Spect50_POP7_Any4Dye for example) with Instrument Protocol on the data collection software. Select conditions as follows.
  DyeSet                      Any4Dye (Z for BigDye)
  Capillary Length            50cm 
  Polymer                     POP6 (to trick the machine)
  Sample Type                 Sequencing Standard 
  Run Module                  Spect50_POP7_1



(3) Click on Edit Param button to open Edit Spectral Param dialog.
(4) Change Minimum Quality Score to 0.92. The default value is 0.80, and it may be OK on the default, but it may cause signal overlap.

Running Standard Samples
Make a plate data for spectral calibration, and run. Check if it is successful on Spectral Viewer. Unsuccessful capillary data is automatically overridden with the next capillary data.



Installing Mobility Files for Autoanalysis
This procedure is unnecessary, if you do not use autoanalysis of UDC but use reanalysis function of SeqA(v.5.1~5.1.1). I recommend to use reanalysis function, if you are concerned about possibilities to destroy the system in use of the mobility file installation procedure.
Combination of the standard ABI3100 basecaller "Basecaller-3100POP6SR.bcp" and mobility files for ABI3730 with POP7 works fine. POP7 mobility for BigDye "DT3730POP7{BDv3}.mob" is preloaded in the mobility folder. Mobility for ET kit "DT3730POP7{ET}.mob" can be downloaded from an Amersham's web site. The installation procedure is as follows:
(1) Make sure to place "DT3730POP7{BDv3}.mob" and "DT3730POP7{ET}.mob" in the folder shown below. Copy, paste and rename these files as "DT3100POP7{BDv3}.mob" and "DT3100POP7{ET}.mob", respectively.



(2) Turn on the electrophoresis machine, and start the UDC. Do not start SeqA.
(3) Run "SeqA_RegisterApp.exe" in the folder shown below.



(4) A DOS window opens, several lines of message appear, and closes.



It is said that the older version of system on WindowsNT recognizes mobility files saved in the appropriate folder. It may thus be unnecessary to install mobilities or reanalyze, but I have not yet tested that.

Electrophoresis Condition
Make an electrophoresis module with an appropriate name (StdSeq50_POP7_1 for example) according to Detwiler et al (2004) with Module Manager. Use StdSeq50_POP6 as a template, and change parameters as follows:
  Oven temperature            60^oC
  Voltage of pre-run and run  8.5kV
  Injection time              40sec
  Data delay time            240sec
  Run time                  6200sec



A span of about 1000bp is read under this condition. Rapid run with a shorter electrophoresis time enables to get 192 sequence reads (two plates) within 24 hours.

Settings for Running Samples
Make an Instrument Protocol with the Protocol Manager of UDC. Click Edit button to call Protocol Editor window and choose DyeSet as Any4Dye (Z for BigDye).




Be sure the DyeSet is correct, or emission signal cannot be separated properly. I recommend to name the protocol as to see IT IS FOR ANY4DYE to run ET samples.
If mobility file is installed for autoanalysis, make Analysis Protocol with Protocol Manager. Click Basecalling tab and choose appropriate basecaller and mobility file. Mobility files for ET kit is incompatible with KB basecaller. Neither BigDye kit is properly basecalled with KB basecaller and renamed KB mobility for 3730 POP7, unlike Detwiler et al (2004).



If installation of mobility files for autoanalysis by "SeqA_RegisterApp.exe" is skipped, use Analysis Protocol for BigDye kit as it is for data collection.
Save the Instrument and Analysis Protocols, and make a Plate Data (sample sheet).
Be sure to choose the Instrument Protocol for Any4Dye (Z for BigDye) in Instrument Protocol1 cell. In Analysis Protocol1 cell, choose Analysis Protocol for ET kit, if mobility files are installed for autoanalysis. Otherwise, choose a dummy Analysis Protocol for, say, BigDye/POP6. Link the Plate Data and start run.

Reanalysis (Mobility Files Only Copied, Not Installed)
After electrophoresis finishes, sample files are saved in the folder according to Result Group assignment. Open these files with SeqA, choose appropriate basecaller and mobility file, and reanalyze.
SeqA(v.5.1~5.1.1) recognizes mobility files, if they are copied to the appropriate folder. Change DyeSet (mobility) and analyze.


Electropherogram of ET kit of 1/20 economy reaction with 50cm capillary + POP7. While many of ET mobility files do not show very good performances, POP5 and POP7 mobilities (especially POP7) are rather satisfactory.


Electropherogram of BigDye kit of 1/40 economy reaction with 50cm capillary and POP7.
Some compression occurs around initial 10~20bases upon electrophoresis condition shown here. An example data of ABI3130/POP7 posted on an ABI web site has also compression. It may be the general tendency of 50cm capillary + POP7.

Data Quality (Phred Scores)



Phred scores of BigDye (left) and ET (right) samples are plotted against read length. Scores on both kits keep more than 20 on averages up to about 900bases, though the leading edge of scores on ET kit is weaker. If sequences near the primer annealing sites are important, use of BigDye kit is better, while ET kit is better to read plasmid inserts with some leader sequences because it is cheaper.

The Leak Problem
POP7 is a low density polymer as expected from its rapid electrophoresis. It is accordingly less viscous and leaky from the injection syringe, though the reserver syringe have never leaked. An ABI web site says the use of POP7/3730 on different machines causes syringe failure and early capillary degradation. Leaked polymer sticks the plunger and the syringe will gradually be clogged to stop processing.


Leakage observed on a syringe after 12 runs (two plates).
Total usage of this syringe is about 300runs.

Polymer sediment at the plunger bar particularly causes clogging, not sediment at the barrel opening. If unfortunately it is clogged, screw the injection syringe off from the polymer block, dip it in water for a couple of minutes to wet the sediment, carefully wipe the sediment away from the syringe, refill polymer and set it debubbling again onto the block. To avoid clogging, carefully wipe polymer sediment away from the plunger with wet Kimwipe after every couple of plates processed or every week intervals.


Wipe polymer sediment with wet Kimwipe.
Be carefull not to overlook tiny polymer sediment at the other side of the plunger.
Do not rotate the plunger.

While the plunger stops at various position at the end of run, it is unnecessary to pull the puluger off from the barrel to clean completely. Polymer leaked in the barrel is wet and would not stick to the plunger.


A portion of the plunger out of barrel and the barrel opening are cleaned.
Then a couple of plates can be processed.

On the other hand, I have not observed early degradation of capillary with POP7 after many runs of fragment analysis (direct loading of dirty PCR products, upon dilution though). I saw rather earlier capillary degradation with POP6 upon runs of dilute PCR products.

Recently I have noticed that a small amount of milli-Q water (20-50uL) applied in the syringe barrel reduces leakage. It can delay beginning of the leakage and also reduce leakage out of some old syringes which have leaked already (added on Dec 19, 2007).



Leakage after one plate run of a syringe to which barrel 20uL water was applied before run (arrow). Total usage of this syringe is more than 400runs.

Reference
Detwiler MM, Hamp TJ, Kazim AL. 2004. DNA sequencing using the liquid polumer POP-7^TM on an ABI PRISM^(R)3100 genetic analyzer. BioTechniques 36:932-933.