Example
Our example probe library consists of 41 x 384-well plates. Plates 1 and 3-41 are full. The last 4x12 block of 48 wells in plate 2 is empty. The controls are in plates 1 and 2.
See figure 1, for a definition of a sub-grid, meta-grid, and source visit (Fig. 1).
First run
We must enter the following command to start the first run of SimArray:
richard@flychip> SimArray.pl -i index.sa
Probe Number
We want to print 15744 wells (384-wells per plate x 41 plates). The empty wells in plate 2 must be excluded from the final array design when we edit the source visit list. If the empty wells had been in the last block of wells in the last plate, we would have entered 15696.
====================================================================== #PROBE NUMBER Enter number of probes (i.e. wells): 15744 ======================================================================
SimArray has saved our response and we can now continue.
Plate format
Our probes are stored in 384-well microtitre well plates.
====================================================================== #PLATE FORMAT Please select a plate format from the list below: ....................................................... Key Plate ....................................................... 0 96 1 384 2 1536 ....................................................... Enter key for the required plate format: 1 ======================================================================
SimArray has saved our response and we can now continue.
Tools available
We want to use a 4x12 tool because we do not want to print the empty wells in plate two. Use of a 4x12 print tool also reduces the number of source visits. Therefore making the print run faster and reducing the required spot density. The estimated maximum meta-grid area is 18 x 54 mm.
====================================================================== #TOOLS AVAILABLE Please select a tool from the list below: ....................................................... Key Tool Pins(X) Pins(Y) Grids Max Area(mm) ....................................................... 0 1x1 1 1 1 4.5 x 4.5 1 2x2 2 2 4 9 x 9 2 4x4 4 4 16 18 x 18 3 4x6 4 6 24 18 x 27 4 4x8 4 8 32 18 x 36 5 4x12 4 12 48 18 x 54 ....................................................... Enter key for the required tool: 5 ======================================================================
SimArray has saved our response and we can now continue.
Source list
We want to print 4 copies of each probe in plate 1. We want to print 4 copies of all probes in plate 2, except for the last 48, where we want 6 copies. The empty wells in plate 2 can be omitted by setting the replicate number to 0. We want to print at least 3 blanks per sub-grid and so we set the source 0 replicate number to 3.
====================================================================== #SOURCE VISITS: Now open sources.txt and amend before re-running this script. Delete or rename sources.txt to repeat this first run. ======================================================================
Example files:
Since we have edited the file, we can now continue.
Second run
We must enter the following command to start the first run of SimArray:
richard@flychip> SimArray.pl -i index.sa
(We want to the source visit map to be space-separated.)
Required spot density
SimArray has counted how many spots we need to print per sub-grid.
====================================================================== #REQUIRED SPOT `DENSITY' Total spots per sub-grid: 377 ======================================================================
This will be of help to us with the next step.
Pins available
We want to select the `Type3' spotting pins for use with the `Type1' robotic spotter.
====================================================================== #PINS AVAILABLE Please select a pin from the list below: ....................................................... Key Pin Arrayer SpotsX SpotsY Total Comment ....................................................... 0 Type1 Type1 40 40 1600 Example 1 Type2 Type1 30 30 900 Example 2 Type3 Type1 20 20 400 Example ....................................................... Enter the key for the required pin: 2 ======================================================================
SimArray has saved our response and we can now continue.
Evaluate pin selection
SimArray has confirmed that the pin type we selected is OK.
====================================================================== #EVALUATE PIN SELECTION Type3 is OK to use because you need to print 377 spots per sub-grid and Type3 can print 400 spots per sub-grid. ======================================================================
No problems here, we can continue.
Compute spots_x and spots_y
To reduce the number of options available SimArray will only display SpotsX and SpotsY combinations that fall between a pre-defined minimum and maximum spot number. SimArray takes the target spot number per sub-grid and used this as the minimum. The maximum limit is the target spot number per sub-grid plus the 'spot number margin'.
We set the maximum number of spots to be printed per to 377 by entering a 'spot number margin' of 10. SimArray then generated a list of options, from which we selected the first by entering the index key 0. We will thus be printing 19 x 20 spots per sub-grid (including 6 blanks).
====================================================================== #COMPUTE SPOTS_X AND SPOTS_Y Enter `spot number margin' (0 for no margin): 10 ....................................................... Key SpotsX SpotsY Total ....................................................... 0 19 20 380 1 20 19 380 ....................................................... Required: 377 ; Min: 377 ; Max: 387 ....................................................... Enter the key for the desired grid layout: 0 ======================================================================
SimArray has saved our response and we can now continue.
Compute print time
Our library is dissolved in 150 mM sodium phosphate buffer and we are using the Type1 spotter. We selected the appropriate wash condition and SimArray has told us that it will take 10.6 hours to print this microarray.
====================================================================== #COMPUTE PRINT TIME Please select a wash condition from the list below: ....................................................... Key Buffer Arrayer Mins Comment ....................................................... 0 Salt Type1 1.7 Example 1 DMSO Type1 1.4 Example 2 Salt Type2 1.5 Example 3 DMSO Type2 1.5 Example ....................................................... Enter the key for the required wash condition: 0 This print-run will take 10.6 hours (approx). ======================================================================
SimArray has saved our response and we can now continue.
Summary report
SimArray has generated a report that includes the above responses and its output.
====================================================================== #SUMMARY REPORT A report has been generated to summarise this print design. If you repeat the second run multiple times, the report will contain details for all of these microarray designs. ======================================================================
The report itself:
We can now take this randomised spot layout and either upload this to the robotic spotter or enter the design manually. Other pin configurations, replicate numbers, slide numbers, and instrument configurations can also be modelled by SimArray, subject to availability of appropriately annotated configuration files. We could then repeat the above process, whilst evaluating a range of different conditions.
