1. The Problem
Well-to-well contamination causes one well on a fingerprinting plate to contain
material from a neighboring well. The contaminated fingerprints
then have false overlaps,
leading to incorrect contig formation and often ruining the assembly. It is therefore
crucial to remove this contamination before assembling.
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.
2. Screening for contaminated wells
To screen for well-to-well contamination, one looks for overlapping plate neighbors.
The cutoff should be set to the same cutoff you will use for the initial assembly.
All suspicious pairs at this cutoff are discarded from the project, before assembly.
In order for FPC to identify neighboring wells, the clones must follow a naming convention, as follows:
name = [library][plate number][row][column]
example: a0370B02
Each field must have a constant number of characters. For example if your plate numbers run from
1 to 1000, they must all be padded to 4 digits, as in the example above. Likewise, all library
tags must have the same number of characters. The row must always be a single letter A-Z, while the
column must always be a two-digit number, e.g. '03' or '18'.
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. It uses 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 add a remark to these clones
and then remove them from the project ("Cancel" them). The Cancelled clones can
be restored after the assembly, if it is desired to analyze them further.
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