Scripts and reagent cassette modifications needed to obtain longer reads on the 454/Roche GS-FLX without the XLR reagent update

Doug White and Graham Wiley

Roe Lab, University of Oklahoma

September 20, 2008

Installing the 126x FLX run scripts

1. The 126x run script is available here

2. Upload the expanded .icl run scripts on to the FLX computer into the directory:

usr/local/rig/runScripts/70x75/TACG/

that contains the default 42x and 100x .icl run scripts.

3. These new .icl files do not show up in the run program:

usr/local/rig/config/gssequencer.xml

4. To run them, start the sequencing run as usual and select the default 100 cycle run script, 100x_TACG_70x75_LR70KIT.icl.

5. Continue as usual until you get to the "Insert new PTP" screen. At the bottom of the window, the blue, active link "Script" followed by the script name, should be highlighted.

6. Click on "Script" and a small window will open showing the contents of the runScripts directory.

a. Select "70x75 and then "TACG" to see all of the .icl files

b. Select the 126x_TACG_DDW.icl run script and click OK.

c. Start the sequencing run.

7. By the way, this is the same way to select the "service" directory to run a volume verification or one of the pump calibration scripts.

Reagent cassette modifications, brief protocol:

1. Before adding the DTT to the four 1L buffer bottles, add 2.5ml of Buffer CB to each of the Substrate, Inhibitor, and Apyrase tubes.

2. Add 174ul of apyrase (instead of 164ul) to the apyrase tube to maintain the original concentration.

3. Transfer 100ml from the buffer3 bottle to the buffer0 bottle.

Reagent cassette modifications, detailed protocol:

Before beginning the protocols described below, complete the preparation of the picotiter and the pre-wash run as described by Roche. Then, follow the buffer thawing and cassette loading below, noting that only steps 7, 8, 11, 18 and 19 indicated in bold italic, differ from those described in the Roche manual

1. Once the reagents in the reagent tube-tray have been thawed (we thaw them in the sink), load it into the 454/Roche GS-FLX, along with the tube of apyrase, 2 tubes of dATP, and 4 tubes of DTT.

2. Remove the four 1L bottles of Buffer CB from their box.

3. Unlock the camera door.

4. Remove the pre-wash cassette from the reagent bay.

5. Unscrew the caps on the reagent tube-tray and the Buffer CB bottles.

6. Place the open bottles of Buffer CB and the reagent tube-tray in the reagent cassette.

7. Transfer 2.5mL from one of the Buffer CB bottles to the Substrate, Inhibitor, and Apyrase tubes respectively in the reagent tube-tray

8. Add 174ul (as opposed to 164ul) from the tube of apyrase to the apyrase buffer tube in the reagent tube-tray.

9. Add 1.5ml from the 2 tubes of dATP to the dATP buffer tube in the reagent tube-tray

10. Add 1ml DTT from the 4 tubes of DTT to the 4 bottles of Buffer CB.

11. Transfer 150ml Buffer CB from bottle 3 (at the back of the cassette) to bottle 0 (at the front of the cassette).

12. Place the reagent cassette in the instrument.

13. Remove the previous runs' plate from the camera door.

14. Clean the plate area in the camera door with EtOH and a kimwipe

15. Clean the camera lens with a lens-cloth.

16. Load the new plate into the camera door.

17. Set up the run parameters by clicking "Run" on the instrument software control panel, following the popup window menu/selections.

18. On the final window, just before hitting start, there will be line with a hyperlink called "scripts" next to a file name showing the script that will be run. Click on the hyperlink "scripts" to bring up a file menu. Navigate to the 130x script by opening the folder 70x75 and then TACG. Highlight the 130x script and hit choose.

19. The hyperlink "scripts" in the last run parameters window will now have the 130x file shown next to it. Hit start.

20. Wait until the instrument shows a blinking green light and the software indicates it is running.