Well-to-well contamination causes one well on a fingerprinting plate to contain material from a neighboring well. The contaminated wells give rise to chimeric fingerprints containing bands from two different clones. The contaminated clones then falsely overlap with their contaminant clones, leading to incorrect contig formation, which is very difficult to eliminate once it has occurred; hence, well-to-well contamination must be removed before the assembly is attempted.

2. Screening for contaminated wells

We emphasize that it is well-to-well contamination which causes the major problems in assembly. Contamination by extraneous material, e.g. organelles, causes one or two obviously bad contigs, but does not undermine the assembly as a whole.

To screen for well-to-well contamination, one examines pairs of plate-neighbor fingerprints looking for ones that overlap, using a given cutoff threshold; a value of 1e-50 seems to work well for HICF. All pairs overlapping to this threshold are discarded from the project, before assembly.

Identification of neighboring wells requires that clones adhere to a naming convention; otherwise, the well origin cannot be identified. Therefore, decontamination should take place before any rearraying.

3. The FPC decontamination function

The function is found on the Clone Search menu. On the main window, press "Clones" button, and then the "Search Commands" button. The "Contaminated" search option is at the bottom of the menu.

Clicking the menu item twice causes it to run and execute the screen described above, using the cutoff and tolerance which have been entered into the Main Analysis window. When it is done, the suspicous clones are displayed in a keyset. Right-clicking on the keyset, one can remark these clones, move then to a certain contig, or remove them from the project ("Cancel" them). In order to have them available for later analysis, the best procedure is to
A. Add a remark to them so they can always be found
B. Move them all to contig 1, using the right-click "move to ctgN" function
C. Run the assembly, making sure to skip these clones by setting "Contig size <= 1" next to the "Kill" button on the Main Analysis page
D. When the assembly is done, move all the contaminated clones to ctg0.

Now the suspicious clones are all singletons, with remarks so they can be studied later, if desired. For example, one may have an important marker on it from laboratory work. Clones can be located on the map later, without disrupting the assembly, using the "Keyset-->FPC" function on Main Analysis.