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MTP tutorial | ||
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Written by
Fred Engler Aug 2003
ContentsA. IntroductionB. MTP using Fingerprints 1. Finding overlapping clone pairs using fingerprints (Step1) 2. Viewing overlapping clone pairs in the contig display(Step3) 3. Picking MTP clones(Step2) 4. Viewing MTP clones in the contig display C. MTP using Fingerprints and draft sequence alignments to BES D. Saving MTP results E. Mandatory clones F. Split BSS Contigs G. HICF Contigs Also see: MTP simulation results A. Introduction Back to topSelecting an MTP (Minimal Tiling Path) is the task of picking a set of minimally overlapping clones that span an entire contig. Due to inexact coordinates in the CB (Consensus Bands) map, one cannot pick overlapping clones based solely on their position on the map. There are two methods using two different input sources for picking MTP clones: 1) the fingerprint method, in which overlaps are determined by looking at the clone fingerprints and their map position, and 2) the BSS-draft method, in which sequence comparison between draft sequence and BESs (BAC End Sequences) via BSS is used. The first method involves analyzing the fingerprints of a pair of overlapping clones for shared restriction fragments (bands), and verifying the integrity of the fingerprints of the potentially overlapping pair by matching bands with a spanning and two flanking clones. In the second method, map overlap is confirmed by a draft sequence contig matching two BESs of the overlapping clone pair. The first method uses information that is already present in the physical map, but the overlaps can be inexact. The second method requires sequence information, but gives very exact overlaps. The 'Select MTP' function can use fingerprints, BES draft sequence comparison results, or both as input for picking MTP clones. When both are used, precedence is given to overlaps verified by the BSS-draft method. There are two steps in automatically picking MTP clones: 1) finding a set of overlapping clone pairs, and 2) picking a contiguous path of overlapping clone pairs through a contig. The following sections guide you through an example of using the automatic MTP picking function of FPC. Before you begin, please download the files used in this demo by clicking here. Uncompress the files:tar xzvf mtpdemo.tar.gzChange directory to the mtpdemo directory: cd mtpdemoNext, start FPC with the demo file by typing fpc mtpdemoon the command line. Open the window for selecting MTP clones by clicking on the 'MTP' button from the Main Menu. The following window appears:  B. MTP using Fingerprints1. Finding overlapping clone pairs using fingerprints Back to topThis step locates all pairs of overlapping clones that satisfy the criteria given in the top part of the window. No sequence information is used in this step. The fingerprint bands and the map positions of clones are used in the analysis.Make sure the 'Use fingerprints' options are turned 'on' (the circle is filled). Leave all parameters at their default value. An explanation of the parameters is given in the online help, which may be read by clicking on the 'Help' button at the top of the window. Click on the 'Find overlapping pairs' button. This starts the computation process. You will see on the standard output the progress being made through the contigs, as shown below: ********** Find overlapping pairs *********** Read 114098 bands from .cor file. Band range(720 3298). Find Fingerprints Pairs Clone pairs for ctg1 (clones 314)...3837 pairs Clone pairs for ctg2 (clones 221)...2880 pairs Clone pairs for ctg3 (clones 92)...1016 pairs . . . Clone pairs for ctg40 (clones 5)...1 pairs Clone pairs for ctg41 (clones 2)...0 pairs Contigs with zero pairs 2 Identified 33456 fingerprint pairs // All contigs Min FPC overlap 0 Max FPC overlap 20 // Use Fingerprints: Min Shared Bands 6 ********** Finish overlapping pairs ***********When the computation is complete, the button will turn gray. 2. Viewing overlapping clone pairs in the contig display Back to topWe will come back to STEP 2, but first we will look at the results from STEP 1 by going to STEP 3 (which shows results from both steps). To see the overlapping pairs in the contig display, select a contig via the 'Contig' text box, and click the 'Next' button beside 'Step through pairs'. The selected contig will open, and the first pair, along with the spanning and flanking clones, will be highlighted. As you continue to click on 'Next', each pair will in turn be highlighted: If the 'Show fingerprints (fp only)' option is turned on, the Fingerprint window will also open, showing a series of fingerprints:  A total of five fingerprints will be shown, and five clones are highlighted. In the contig display, the clones highlighted in blue indicate the clone pair. The pale blue clone spanning the overlap of the pair (called the spanner) verifies the shared bands of the pair. Extending to the left and right of the pair are two clones highlighted in gray. These clones confirm bands in the pair that are not confirmed by the spanning clone. The Fingerprint window is used to show how bands are shared. The following color scheme is used: Cyan -- band is shared by both clones in the pair and the spanning clone. Green -- band is shared only by the left clone in the pair and spanning clone. Blue -- band is shared only by the right clone in the pair and spanning clone. Violet -- band is shared by a clone in the pair and its flanking clone, but not by the spanner. Red -- band in a pair or spanning clone that is unconfirmed; a mismatch. In the standard output, information on the shared bands and unmatched bands, along with the length of the pair clones, is given:
Fingerprint pair:
L-flank Left Spanner Right R-flank
z2598 z2597 z2602 z2612 z2611
122880 167936 (length)
6 10 12 20 9 (shared)
- 2 1 0 - (mismatch)
The numbers displayed on
the terminal for each pair have the following
correspondence to the colors of bands in the Fingerprint
window.
You may get output as follows:
Fingerprint pair: olap 49152
L-flank Left Spanner Right R-flank
z2598 z2597 z2602 z2612 z2611
122880 167936 (length)
6 10 12 20 9 (shared)
- 2 1 0 - (mismatch)
which indicates that no valid spanner and flankers could be found, though the pair
does qualify based on the user input.
 To revert to the full-sized window, click on the 'Full' button. 3. Picking MTP clones Back to topNow we will go back to STEP2 to automatically select the MTP. Using the shortest paths algorithm, a minimal path of clones is picked through a contig based on the amount of clone overlap (shared bands) and clone size. To run this, click on the 'Pick MTP clones' button in STEP 2. The following text will be displayed:
************ Starting PickMTP ************
Building graphs completed.
Finding MTP completed.
Average MTP clone size: 143959
Contig totals: (in CB units)
Contig Ctg len # MTP overlap # of gaps gap length %covered
------- ------- ----- ------- --------- ---------- --------
ctg1 493 14 102 0 0 94%
ctg2 303 8 62 0 0 94%
ctg3 159 4 23 0 0 92%
ctg4 137 3 16 0 0 79%
ctg5 185 5 30 0 0 94%
ctg6 538 17 133 0 0 97%
ctg7 298 11 76 0 0 94%
ctg8 872 26 184 0 0 97%
ctg9 257 7 48 0 0 89%
ctg10 100 3 16 0 0 95%
...
ctg40 37 1 0 0 0 64%
ctg41 41 1 0 0 0 70%
Clone overlap (base pairs):
Positive:
20000- 30000- 40000- 50000- 60000- 70000- 80000-
29999 39999 49999 59999 69999 79999 89999
80 31 21 7 5 2 3
Total positive overlap: 5177344
Average positive overlap: 34747
Number of positive clone overlaps: 149
Clones picked:190
BSS pairs: 0 (0%)
Fingerprint pairs: 149 (98%)
Single MTP clones: 3
Mandatory clone pairs: 0 (0%)
Expressway junctions: 0 (0%)
Number of clones in MTP: 190
Number of mandatory clones: 0
Total gap span: 0 kb
Total MTP span: 24019 kb
Percent of map covered: 92%
// All contigs Prefer large Mandatory:
************ Finished PickMTP ************
The 'Pick MTP clones' button will turn gray once the
process completes.
In the table displayed on the standard output, the "ctg len" column displays the length of the contig, and the "overlap" column displays the total overlap of the clones in the MTP of the given contig, both lengths being given in FPC units. Gaps in the MTP are counted wherever there is a break in the MTP (not including pairs with a negative overlap, which can happen with BSS pairs), where this number does not include any potential uncovered segments of the contig beyond the ends of the MTP. Thus "# gaps" displays the number of such breaks in the MTP, and "gap len" is the total length of those gaps in FPC units. Gaps should usually be 0 if fingerprint data is used, because there is almost always at least one viable path through the contig; however, if only BSS data is used, or if the parameters are set very stringently, then gaps may appear. We emphasize that these gaps are relative to the FPC contig; typically, additional gaps will be found when sequencing is performed, because the FPC contig embodies only partial information about the underlying sequence. Finally, "%covered" displays the fraction of the contig between the ends of the MTP on that contig (excluding gaps). Note that all of the lengths given in the MTP report to the standard output are based on the numbers of bands and the average band size in the FPC project, and are therefore only approximations of the actual overlaps, gaps, etc. Note: If you change the pair parameters, you will need to first rerun the 'Find overlapping pairs', and then rerun the 'Pick MTP clones' function. 4. Viewing MTP clones in the contig display Back to topThe MTP clones are viewed in the contig display the same way as the pairs, using the 'Next' and 'Previous' buttons beside 'Step through MTP'. Sometimes a complete path cannot be found through a contig. The contiguous paths are called "expressways". Look at the information in the standard output to see how the clones make up the expressways. For example,
Clones (1, 2) in expressway of 14:
Fingerprint pair:
L-flank Left Spanner Right R-flank
z2598 z2602 z2610 z2628 z2629
176128 126976 (length)
8 28 7 6 18 (shared)
- 0 0 0 - (mismatch)
tells us that we are looking at the 1st and 2nd
clones in a contiguous path of 14 clones. Click on the
'All' button to see all picked clones highlighted in blue.
Look at the text output to see where junctions occur.
Whenever a path or expressway does not span the entire contig, a new expressway must start near the last clone of the previous expressway. The process of choosing a clone to begin the new expressway can only be based on overlaps of clones on the CB map, and should therefore be verified by a person. FPC will display messages to the standard output signifying such regions of "weak overlap" whenever they occur in the MTP (see the section on picking an MTP using BSS pairs, below, for an example). C. Adding overlap data from draft sequence alignments to BES
A much more accurate estimate of the overlap of two clones can
be obtained if a BES from each clone hits a particular
draft sequence contig. If your species has both draft sequence and BES,
it is recommended to use this data in addition to the
fingerprint overlap data.
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