Contamination Check

There are two kinds of potential contaminations during BAC library construction, filter duplication and fingerprinting:

• cross-contamination between adjacent clones and of nonadjacent clones within plates and
• chloroplast DNA contamination in plant BAC libraries.

Chloroplast DNA contamination

In the plant BAC library construction, some clones contain chloroplast DNA inserts not nucleus DNA. Usually clones containing chloroplast DNA account for 0.5% –1.5% in BAC libraries (Tomkins et al. 1999, Yu et al. 2000). There are two approaches to check the clones containing chloroplast DNA. One is the hybridization. The other is the program screening developed in GenoProfiler.  In GenoProfiler, the screening of clones containing chloroplast DNA is performed by comparing the similarity between chloroplast DNA and BAC clone fingerprints.   A match percentage, representing similarity of fingerprints, is defined as match bands / min(bands of chloroplast DNA fingerprints, bands of  a clone ) * 100.  Two bands match if the difference of two band sizes is less than or equal to a user-defined tolerance (default 0.4 bp).  If the match percentage of fingerprints is very high and greater than a user-defined threshold, we may infer that the clone contains chloroplast DNA.

Chloroplast DNA fingerprint could be obtained from simulating digestion of the sequenced chloroplast DNA by using the same BAC fingerprinting procedure (Luo et al. 2003).  Because of discrepancy between the predicted sizes in simulating digestion and fragment sizes in capillary electrophoresis, we can employ a direct approach to obtain chloroplast DNA fingerprint. First we can detect some chloroplast DNA-contaminated clones using hybridization of chloroplast DNA as marker with high-intensity filters of BAC clones, and then get the fingerprint of the chloroplast DNA using fragment size frequency analysis. In the wheat D genome physical mapping project, the hybridization experiments using wheat chloroplast DNA specific probes (a pool of rbcL and petD) detected 76 positive clones with wheat chloroplast DNA. Comparison of the fragments of those clones showed that they had very high similarity (match percentages > 90 %) and hence were inferred as real chloroplast DNA-contaminated clones. According to the fragment size frequency analysis of the 76 chloroplast DNA-contaminated clones, 112 fragments (36 for blue color, 28 for green, 34 for yellow and 14 for red) with fragment frequency > 80 % were chosen as wheat chloroplast DNA fingerprint (data not shown). This fingerprint is used as screening wheat chloroplast DNA-contaminated clones.

Cross-contamination

In a plate arrayed with random clones, it should be a rare event that two adjacent or nonadjacent clones have high level of similarity of clone fingerprints. If this event happened, we may infer that two clones are cross-contaminated each other.  In GenoProfiler, we take into account three potential cross-contamination sources during filter making, duplication and fingerprinting:

• cross-contamination of adjacent clones within 384-well plates, 
• cross-contamination of adjacent clones within 96-well plates transferred from 384-well plates in fingerprinting and 
• cross-contamination of both adjacent and nonadjacent clones within 384-well plates or 96-well plates with very high similarity between clone fingerprints (simply called as high overlapping check).

The same strategy of comparing the similarity between two clone fingerprints as in chloroplast DNA contamination check is used to screen cross-contamination clones.  The match percentage here is defined as match bands / ((bands of clone 1+bands of clone 2)/2) * 100. If the match percentage of fingerprints is very high and greater than a user-defined threshold, we may infer that two clones are potentially cross-contaminated. In cases 1 and 2 above, only clone fingerprints between directly adjacent cells are compared. In case 3, however, all pair-wise comparisons in a plate will be performed.   Thus, contamination check of the case 3 will include partial or whole check results of cases 1 and 2 depending on if the same or different match percentage thresholds are set. The high overlapping check is primarily designed to screen clones with high similarity of fingerprints in which some of them may be due to cross-contamination, however, most of them have real high similarity.  Thus, as default, the match percentage threshold is set to 80% for high overlapping check and to 70% for other two cases.

This module will detect all 4 potential contamination sources by comparing the fingerprinting pattern similarities between  clones or between clone and chloroplast DNA fingerprint pattern. If match percentage of fingerprints is very high and greater than a user-defined threshold, we may think there is high possibility that two clones are cross-contaminated or a clone is contaminated by chloroplast DNA.

Steps of using this module:

1. Set clone naming policy: see Sample File and Clone Naming and Sample File and Clone Name Setting .

   • Click Setting menu, choose Clone/Sample File Naming menu item:

     

   • Set appropriate parameters and click Save, Cancel or Ok button.
     Note: This is very important step for correct contamination check. Before you do contamination check, you need to correctly specify  clone names in the size files.
2. Set contamination check parameters: See Contamination Check Setting. 
   • Click Setting menu, choose Contamination Check menu item:

     

   • Set appropriate parameters and click Save, Cancel or Ok button.
     
3. Choose contamination source: Four potential contamination checks are available. You may decide which contaminations potentially exist in you data. You may choose just one each time and get clone list of different contamination sources. If you check all four contamination sources, you will get a clone list of all contamination sources together. If you want to check chloroplast contamination, you must have a chloroplast DNA fingerprints available.
   • 384-Well Plate Check: Check the possible contaminations of adjacent clones between vertical and horizontal wells. If two adjacent clones are highly matched in fingerprints and march percentage is greater than the preset threshold, we put two clones to possible contaminated clone list.
   • 96-Well Plate Check: When transferring clones from a 384-well plate to 96-well plates, four 96-well plates will be created. There is possibility of cross-contamination in 4 of 96-well plates. The same strategy as 384-well plate check is used for 4 of separate 96-well plates.
   • High profile sharing Check: Check the possible contaminations of nonadjacent clones within 384-well plates. Program will pair-wisely compare the fingerprints of each pair of clones . This check actually includes both 384 well plate check and 96 well check if you set the same thresholds. If there is too high match percentage of fingerprints between two nonadjacent clones, there will be high probability to say these two clones have contamination problem. Note: The clones with high profile sharing in a plate are not necessarily contaminated clones. so you need to set a higher threshold.  
   • Chloroplast DNA Check: Check possible chloroplast DNA contamination.
4. FPC Size File Directory : Type or choose a directory which includes FPC size files. The size files can be in any subdirectories.
5. Chloroplast Fragment Size File: Type or choose chloroplast fragment size file. Chloroplast DNA hybridization can provide a set of clones which have positive reactions to chloroplast DNA. From the fingerprints of those clones, you can create a chloroplast DNA fragment size file. (See Vector, Repeat, and  Chloroplast Band Size Files).
6. FPC Size Clone Name Filter Pattern: If you want to process all clones in the specified directory, just type "*" in the text field. You can write a regular pattern list to tell program process only the clones which names match the patterns. For example, if you want to process clones with library code "RI" or plate number "034", you may write regular expressions as *RI* and *034*. In the text field, you need type *RI*, *034* . A comma "," must be inserted between two patterns. Please see Appendix B: Wild Card Pattern.
7. Tolerance For Band Matching: A tolerance is size limits within which the observed sizes of two restriction fragments must be to be considered identical fragments during contig assembly by the FPC program. The setting of tolerance is limited by variation in fragment sizing across time and instruments. The same tolerance as FPC contig assembly is used here to check vector bands.
   
8. If all settings are Ok, click Start button. You may click Cancel to stop the submitted job.
9. View contamination patterns of each library+plate. Different colors in wells will show different kinds of contamination sources.
   

10. Save the potential  contaminated clone list: After the job has finished, a log window will show you a clone list. You may view the contamination patterns of each plate, and then you may Click  button on tool bar or Click File menu, choose Save or Save As menu item to save clone list as a text file.