User`s Manual for FISH (Fast Identification of Segmental
Contents
1. 3 3 2 44 KKK KKK 2 KKKXx 1 1 KKKXx 0 SC 1 2 1 1 1 1 CHAPTER 5 AN EXAMPLE KK A KK A KKKKKKKKKKKKKKKKKKKKKKKKKKKIG KKKKKKKKKKKKKKDD KKKKKTS 2 2 5 5 A CONTIG OUTPUT FILE 29 66 0 00e 00 SAL 79 0 00e 00 p 1 0 10 20 30 40 50 5 5 A contig output file The opening lines of the file contigs txt are shown here Fri Jun 27 11 15 44 2003 FISH v1 0 FISH is copyright c 2003 University of North Carolina at Chapel Hill Authors Sugata Chakravarty and Todd J Vision parameters MIN_SCORE 200 MAX_DIST 10 contigs contig markers features 1 6494 5913 2 4038 3711 3 5221 4777 4 3825 3467 5 5888 5453 features feature contig orientation genes 0 1 1 At1901010 30 CHAPTER 5 AN EXAMPLE 130102 190103 190104 190105 190106 107 108 12. The scores shown are bit scores from TBLASTX Note that with a MIN_SCORE of 200 all but the last match shown would be discarded At1g01030 At1g25560 112 At1g01030 At1g50680 73 At1g01030 At1g51120 72 At1g01030 At1g35240 63 At1g01030 At1g35520 60 At1g01030 Atlg77850 58 At1g01030 At1g34310 58 At1g01060 At1g18330 133 At1g01060 At1g01520 109 At1g01070 At1g11450 313 5 4 An example of captured STDOUT By using the redirection operator the output that FISH writes by default to the terminal is captured to a text file In the sample output this file is named fish_out txt 26 CHAPTER 5 AN EXAMPLE Fri Jun 27 11 15 47 2003 FISH v1 0 FISH is copyright 2003 University of North Carolina at Chapel Hill Authors Sugata Chakravarty and Todd J Vision Control File control txt processing contigs min_score 200 max_dist 10 contig msrkers features 1 6494 5913 2 4038 JLL 3 5221 4777 4 3825 3467 5 5888 5453 writing contig output to contigs txt processing matches min score 200 top hits 5 contigl contig2 points cells 1 1 2143 17478828 1 2 2018 21943144 1 3 2088 28246400 1 4 1538 20500372 1 5 1986 32243588 2 2 TSI 6883905 2 3 1460 17727448 2 4 1239 12866037 2 5 1164 20236084 3 3 849 11407476 3 4 1300 16561859 5 4 AN EXAMPLE OF CAPTURED STDOUT 3 5 1977 26048980 4 4 711 6008311 4 5 1551 18905552 5 5 1293 14
3. value of the threshold paremeter T the total number of points in the grid the total number of features in the dataset the total number of cells in the grid the proportion of cells that contain a point h the size of the neighborhood dr the minimum block size to be reported and the total number of blocks found in the dataset 22 CHAPTER 4 FILE FORMATS This is followed by individual tables for each block Within each there is an upper panel that lists the number of points and the associated contigs And there is a lower panel that lists each point in the block in sequence For each the table shows the number of the point the distance from the previous point the names of the markers for the associated features in the format markers for feature 1 markers for feature 2 and the product of the orientation of the point with the previous point in the block This product may take a value of 1 0 or 1 If values switch frequently between 1 and 1 then transcriptional orientation is not well conserved within that block and the user may wish to regard this block with suspicion Zero indicates unknown At the bottom of the blocks file the number of blocks seen between each pair of contigs is reported Below that the length distribution of blocks is reported For blocks with a given number of points the observed and expected numbers of blocks are shown followed by the p value for blocks of that length The p value is calculated as descri
4. 02e 03 94e 04 43e 05 15e 07 96e 08 00e 00 00e 00 00e 00 00e 00 00e 00 00e 00 35 Bibliography 1 Calabrese PP Chakravarty S Vision TJ 2003 Fast identification and statistical evaluation of segmental homologies in comparative maps Bioinformatics 19 174 180 2 TJ Vision DG Brown SD Tanksley 2000 The origins of genomic duplications in Arabidopsis Science 290 2114 2117 36
5. 1 1 11 616 237 3 1 1 11 1668 665 4 1 1 11 2146 580 5 1 1 11 4808 582 6 1 1 11 5050 326 7 1 1 13 2412 290 8 1 1 13 2499 303 32 CHAPTER 5 AN EXAMPLE 9 1 1 13 3263 10 1 1 18 5288 11 1 1 30 1288 12 1 1 31 1355 13 1 1 31 1699 14 1 J 41 4440 PS 1 1 49 4531 5 7 A blocks file Selected lines from the file blocks txt are shown here Fri Jun 27 11 15 47 2003 FISH v1 0 FISH is copyright c 2003 University of North Carolina at Chapel Hill Authors Sugata Chakravarty and Todd J Vision Control File control txt parameters T 0 05 total points 22068 total_features 23321 total_cells 2 71923e 08 h 8 11554e 05 d_T 25 6408 Min_Block_Size 3 block_prob 0 001 total_blocks 508 block 0 401 207 588 549 553 242 273 5 7 A BLOCKS FILE points contigl 79 1 point dist 1040 0 1041 1042 1043 1044 1050 1056 1057 1058 11 1061 15 1062 1063 1064 3 CO 0 JU WN E FA block 1 points contigl 30 1 point dist 1246 0 1249 12 1250 14 1251 4 1252 13 1256 1258 1262 1263 1265 1266 33 contig2 1 markers orientation At1g19320 At1g75040 1 At1g19330 At1g75060 1 At1g19350 At1g75080 1 At1g19360 Atlg75110 At1g75120 At1g19370 At1g75140 1 At1g19400 At1g75180 1 At1g1
6. 2 The executable will be located in C lt DESTRED DIRECTORY gt FISH 1 0 fish exe For Example Unzip to C Program Files From a command line type C Program Files FISH 1 0 fish exe h Don t forget the quotations marks This executes fish and shows the command line options yu RECTORY gt REQUIREMENTS 1 32 MB of RAM 2 Operating system Known to run on Windows XP ME and NT 1 5 3 Installing the MacOSX executable NSTALLATION 1 Unzip FISH 1 0 osx zip 2 The executable will be located in FISH 1 0 fish REQUIREMENTS 1 32 MB of RAM 2 Operating system Known to run on 10 2 x 8 CHAPTER 1 GETTING STARTED 1 5 4 Testing FISH on sample data You may use the sample files to test that the program is working properly From a command line window go to the directory within the distribution where the sample control file control txt is located Type fish then enter and within a few seconds FISH should complete the analysis Chapter 2 How it works The following is a concise overview of FISH A fuller description of the methodology is given in 1 with which we assume the reader is already familiar There are three steps in the analysis Step 1 From markers to features Step 2 From features to grid Step 3 From grid to blocks Each genome to be compared consists of one or more linear
7. License whose permissions for other licensees extend to the entire whole and thus to each and every part regardless of who wrote it 4 USER S PROVISION AND OR DISTRIBUTION OF SOURCE CODE You may copy and distribute the Program or a work based on it under Section 2 in object code or executable form under the terms of Sections 1 and 2 above provided that you also accompany it with the complete corresponding machine readable source code which must be distributed under the terms of Sections 1 and 2 above on a medium customarily used for software interchange The source code for a work means the preferred form of the work for making modifications to it For an executable work complete source code means all the source code for all modules it contains plus any associated interface definition files plus the scripts used to control compilation and installation of the 4 CHAPTER 1 GETTING STARTED executable However as a special exception the source code distributed need not include anything that is normally distributed in either source or binary form with the major components compiler kernel and so on of the operating system on which the executable runs unless that component itself accompanies the executable If distribution of executable or object code is made by offering access to copy from a designated place then offering equivalent access to copy the source code from the same place counts as distribution of the source code even th
8. e SE CP AE EA EN 18 42 A O a E 19 4 21 Screen 011611 03 ede Se A an 19 42 2 a 20 423 og os ae 21 DA Block TE ve e enn e a el SR nee 21 4 2 5 Simple block ale ela e ae 4 22 An example 23 5 1 A control input file 4 34 448 2 405 6 6 6 den 544 54 de 23 5 2 A map nput flle sa 24 5 3 EE ON 25 5 4 An example of captured STDOUT 25 5 5 A contig output file hn 6 6 6 A eg A 29 5 6 EIERE Ee ER AA A A 30 5 7 AA 0106165711167 A e er eg 32 Chapter 1 Getting started 1 1 Introduction Genomic regions that have descended from a common ancestral region are said to be segmental homologs FISH is software for the fast identification and statistical evaluation of segmental homologs The software treats each input genome as a string of features A feature may correspond to one marker or sometimes to multiple homologous markers that are very close to one another on a contig Each string of features whether it is a compete chromosome or some smaller physical or genetic linkage group is referred to as a contig Matches are pairwise homologies among markers which along with the position of markers on contigs constitute the primary input data FISH finds blocks of putative segmental homology by identifying paired regions in which multiple matches occur and where the underlying features are in roughly col
9. into another language Hereinafter translation is included without limitation in the term modification Each licensee is addressed as you 1 SCOPE Activities other than copying distribution and modification are not covered by this License they are outside its scope The act of running the Program is not restricted and the output from the Program is covered only 1f its contents constitute a work based on the Program independent of having been made by running the Program Whether that is true depends on what the Program does 2 USER S RIGHTS You may copy and distribute verbatim copies of the Program s source code as you receive it in any medium provided that you keep intact all the notices that refer to this License and to the absence of any warranty give any other recipients of the Program a copy of this License along with the Program and conspicuously and appropriately publish on each copy a disclaimer of warranty and the following statement Copyright 2003 University of North Carolina at Chapel Hill If the code has been modified you also may include the following statement Modified by your institution You may charge a fee for the physical act of transferring a copy and you may at your option offer warranty 1 3 LEGAL INFORMATION 3 protection in exchange for a fee 3 RIGHTS IN DERIVATIVE AND COMBINED WORKS It is not the intent of this section to claim rights or contest your rights to work writt
10. number under the null model then blocks of that size are flagged in the summary output Some of these blocks may well be spurious Chapter 4 File formats 4 1 Input files There are three types of input files control files map files and match files The control file tells FISH the location of the map and the match files 4 1 1 Control file A control file is used to specify the paths of the map and match files The format of the control file can be seen in the sample provided It is divided into two sections one for map files and one for match files At a minimum one map file and its associated match file are required To compare two different contigs map and match files are required for each In addition one or two match files must be specified that list the matches between features in contig 1 vs 2 and or 2 vs 1 A list of the map files is preceded by the optional token maps followed by a newline character followed by a separate line for each contig that is to be analyzed Each of those lines contains an integer followed by whitespace followed by a valid filename path followed by a newline character The integer 1s taken to be the name of a contig and the filename path tells FISH where to find the map file for that contig A list of the match files is preceded with the optional token matches followed 17 18 CHAPTER 4 FILE FORMATS by a newline character followed by a separate line for each pair of
11. pair of contigs For an example see Section 5 There are three columns in the matchfile The first two columns may be strings but the third column needs to be an integer The first and second columns contain the gene names from the first and second contig respectively The integer in the third column must be some measure of the strength of the match between the two markers FISH assumes that the larger the integer the stronger the match If information is not available on the strengths of the matches between genes one can for example assign all scores to be zero and set MIN_SCORE to be zero at runtime All self matches are discarded 4 2 OUTPUT 19 4 2 Output FISH can generate a variety of different output streams depending on the user s selections FISH can also report the time spent at various stages in the analysis This option can be selected by using the t switch see Section 3 4 2 1 Screen output Unless the QUIET MODE has been selected FISH will report results from each stage of the analysis to standard output STDOUT See Section 5 for an excerpt from the sample file fish out txt The STDOUT stream may be directed to a file rather than to the terminal by using the gt redirection operator like so fish options gt myfile txt At the beginning of the process FISH sends STDOUT a brief title message including the current version number the date and time at which the process was initiated and the control f
12. 09 110 H amp amp o Jo N amp o AO 1 WO NH Oo A N A A N N A A DDD DD DD DD DD DD Db DD DD DD GT a q EE CON EE CCE Met et GE GF GP SE E ver ete q EE eh GE ct TEE CO DO CH CH CH CH CH DO CH CH CH CH CH CH OOO CO CH CH CH CH CH CO CH CH Oo CO CO CH CO CH CH CO CO CH CO CO Oo CH Y GA LA A A A A A A A A pas LA LA N N Ww A A OO 5 6 A grids output file The opening lines of the file grids txt are shown here Fri Jun 27 11 15 46 2003 FISH v1 0 FISH is copyright c 2003 University of North Carolina at Chapel Hill Authors Sugata Chakravarty and Todd J Vision 5 6 A GRIDS OUTPUT FILE 31 Control File control txt parameters MIN_SCORE 200 TOP_HITS 5 subgrids contigl contig2 points cells 1 1 2143 17478828 1 2 2018 21943144 1 3 2088 28246400 1 4 1538 20500372 1 5 1986 32243588 2 2 751 6883905 2 3 1460 17727448 2 4 1239 12866037 2 5 1164 20236084 3 3 849 11407476 3 4 1300 16561859 3 5 1977 26048980 4 4 711 6008311 4 5 1551 18905552 5 5 1293 14864878 points point contigl contig2 featl feat2 score 0 1 1 6 954 328 1 1 1 11 300 395 2
13. 864878 writing grid output to grids txt processing blocks T 0 05 total_points 22068 total_features 23321 total_cells 271922816 probability h 0 000081 d_T 25 640781 min_edges 2 block_prob 0 00100 total_blocks 508 contigl contig2 blocks 1 1 50 52 43 34 25 27 48 48 18 15 25 53 16 33 21 aor O1 VS GA OD ds WBN OB W DN Or CO uN NN N writing output to blocks txt 28 block statistics points p value 3 1 00e 00 ia LAS 4 9 22e 01 RAR SOS 5 1 17e 01 6 02e 03 7 94e 04 8 43e 05 9 15e 07 1 96e 08 1 00e 00 A p s ss ps o IS OL Gs wUNHO A LA VO vs GA GA GA NN k k k k k DN y VG OO k vw MOANA OD amp GA b i r OY o N o DO DD O 0 O OO O DO Or Or OO OO NO Or OO OY O db Sy IND ON De De De De De De De De De De De De De De O OO Ch ooo Ch Ch Ch Ch Ch Ch Ch Ch Oo Ch Ch CH CC CC CH CH O OO Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch CH CC CC CH CH O CH Ch Oo Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch CH CC CC CH CH frequency KKKKKKKK KKKKKKKK KKKKKKKK KKKKKKKK 2 KKKXx KKKKKKXK
14. 9450 At1g75220 1 At1g19480 At1g75230 1 At1g19550 At1919570 Atlg75270 At1g19650 At1g75370 1 At1g19660 At1g75380 At1g19680 At1g75400 1 At1g19700 At1g75410 contig2 1 markers orientation At1g22920 At1g71230 1 At1g22970 At1922980 Atlg71150 At1g23010 At1g71040 1 At1g23030 At1g71020 1 At1g23080 At1g70940 1 At1g23110 At1g70900 1 At1g23140 At1g70800 At1g70810 At1g23170 At1g70770 1 At1g23190 At1g70730 1 At1g23210 At1g70710 1 At1g23240 At1g70670 Atlg70680 1 1 ll i 34 CHAPTER 5 AN EXAMPLE 1267 3 At1g23260 Atlg70660 1268 9 At1g23290 At1lg70600 block 507 points contigl contig2 3 5 5 point dist markers orientation 21978 0 AT5g53950 AT5g61430 1 21982 23 AT5g54230 AT5g61420 1 21988 21 AT5g54380 AT5g61350 1 by contig contigl contig2 blocks 1 1 50 1 2 52 1 3 43 1 4 34 1 5 25 2 2 27 2 3 48 2 4 48 2 5 18 3 3 15 3 4 25 3 5 53 4 4 16 4 5 33 5 5 21 by size points obs exp D 5 7 A BLOCKS FILE N OH OO OAD OB GA pa H 44 60 66 79 243 83 35 21 22 OO L GO On k k ADEN On CO CH 23e 01 55e 00 24e 01 04e 03 94e 04 43e 05 97e 07 40e 08 65e 09 05e 11 00e 00 00e 00 00e 00 00e 00 00e 00 22e 01 17e 01 CH On HN O
15. Blocks consist of sequences of points in which each point is within the neighborhood of the previous one FISH calculates the size of the neighborhood for all points based on the total number of points m and cells n in the grid The probability of a cell containing a point is h m n From this and from an adjustable parameter T see 3 1 FISH calculates the size of the neighborhood In its current version FISH measures distance between two points X Y and X Y using the Manhattan distance d X X Y Yj In order to be 12 CHAPTER 2 HOW IT WORKS considered neighbors two points must be closer than 1 log 1 T 1 di A a 2 1 T 9 ol 4 Sr 2 3 Step 3 From grid to blocks FISH uses a dynamic programming algorithm to identify all maximally extended blocks For a detailed description of the algorithm see 1 2 3 1 Choosing among multiple neighbors It can happen that a point may be in the neighborhood of more than one other point In such a case it is necessary to specify rules for which point will be chosen to extend the block To do this FISH ranks the cells within each neighborhood and chooses that neighbor having the highest rank using an ad hoc formula that gives preference to points that preserve the colinear order of matching features along the two contigs The rank r for each cell in the neighborhood of the candidate point is 1 d r w ST 2 2 where n is the number of cells in the point s neighbo
16. User s Manual for FISH Fast Identification of Segmental Homology version 1 0 released 30 June 2003 Copyright 2003 University of North Carolina at Chapel Hill Contents 1 Getting started 141 introduction e Gens rs nw E ee Se E 1 2 Contact information a gas AA AS E 1 35 Legal information 6 6 6 6 6 6 48 late psd die 1 4 Acknowledgements OSs 1 57 Availability ao de ma eek de E e Se we 1 5 1 Installing from source code 1 5 2 Installing the Windows executable 1 5 3 Installing the MacOSX executable 1 5 4 Testing FISH onsampledata 2 How it works 2 1 Step 1 From markers to features 2 2 Step 2 From features to grid 6 6 esses neles a Ba 2 2 1 Removing low ranking matches 2 2 2 Numbering of points 2 2 3 Enforcing symmetry 6 6 6 6 6 6 a ee 4 2 2 4 Defining the neighborhood size 2 3 Step 3 From grid to blocks 2 23 4 lei sais 2 3 1 Choosing among multiple neighbors 2 3 2 Overlapping blocks A toa its ke Ws 6606 ere 3 Parameters 3 1 Algorithmic parameters 479351903000 Ye 6 6 6 oe ale ada 3 2 Input and output parameters 4 File formats 41 Input les e Bata e 5 41 1 Controlfile 17 AND MAD TES rs 18 413 Match files ce se ta o
17. alues of MIN SCORE and MAX DIST This is followed by a table showing the number of markers and features in each contig Finally a table is presented in which for each feature the contig the composite direction and the names of the markers from the map file associated with that feature are shown 4 2 3 Grid file If the PRINT_GRIDS option is selected FISH will output a grid file with the specified name See Section 5 for an example The grid file allows translation between points and features as well a summary of the parameters and results for the match processing step The top of the grid file displays the time at which the process was initiated This 1s followed by the parameter list which includes the name of the control file and the values of MIN SCORE and TOP HITS This is followed by a table showing the number of points and cells for each pair of contigs Finally the points are listed For each point the associated contigs the associated features and the composite score are shown See Section 4 2 4 for an explanation of how the score is derived 4 2 4 Block file If the PRINT_BLOCKS option is selected FISH will output a block file with the specified name See Section 5 for an example The top of the block file displays the time at which the process was initiated and the control file used This is followed by the parameter list This list similar to the one output to STDOUT see 4 2 1 includes the name of the control file the
18. bed in 1 p 7 10 4 1 where rc is the total number of cells in the grid p h nh and n is the number of cells in a neighborhood of size dr 4 2 5 Simple block file FISH provides the option of printing a more computer readable block file by choosing the PRINT_B_SIMPLE option The first two lines report the number of blocks and the total number of points in all blocks respectively Each subsequent line reports the output for one block The first number in each line is the number of points in that block This is followed by comma delimited list of the points themselves Each block is terminated with an asterisk Chapter 5 An example The following truncated input and output files come from a re analysis of the segmental homology within and among the five chromosomes of Arabidopsis thaliana 1 2 All the genes that received AtXgXXXXX codes in the TIGR version 3 0 release of the genome annotation have been included in this dataset Assuming that one is in the same directory as the example input files one can reproduce this session by invoking FISH with the following options fish b blocks txt g grids txt C contigs txt B simpleblocks txt t gt fish out txt An explanation of the content of these files is provided in chapter 4 5 1 A control input file Shown is the complete control txt file The filenames may be entered as paths For example if the map and match files were in a subdirectory na
19. ce Foundation grants DBI 0110069 and DBI 0227314 to TJV and DMS 0102008 to PPC The authors wish to thank Peter Calabrese for his many contributions to the ideas underlying the software 1 5 Availability The latest version of this program along with documentation and examples is available from http ww bio unc edu faculty vision lab FISH FISH is written primarily in C together with C style data input output The source code is designed to be compatible with all versions of gcc GNU C C compiler In addition executables are available for a limited number of platforms The program may require 32 MB of RAM or more at runtime 1 5 1 Installing from source code Installation from source code requires a c compiler the makefile assumes that you have g Fish is known to successfully compile with g 3 1 and 3 2 to see what version of g you have type g v The code is known to compile and run on at least some versions of Solaris 1 5 AVAILABILITY 7 RedHat Linux Windows and MacOSX It should compile and run on all systems with g installed For windows environments MinGW http www mingw org provides a g compiler that works well To install type the following commands 1 tar xzvf FISH 1 0 tar z 2 cd FISH 1 0 3 make all 4 cp bin fish usr local bin may require superuser priviliges 1 5 2 Installing the Windows executable NSTALLATION 1 Unzip FISH 1 0 win zip into MOLA SDESTRED
20. ch feature 2 2 Step 2 From features to grid In order to identify segmental homologies FISH computes a grid for each pair of contigs Points in the grid represent matches between pairs of features The 2 and 2 2 STEP 2 FROM FEATURES TO GRID 11 j coordinates of a point are taken to be the positions of the features within their respective contigs Each position in the grid whether or not a point is present is called as a cell 2 2 1 Removing low ranking matches The number of matches for each feature in the final grid can be limited by an adjustable parameter TOP_HITS If a match is not one of the highest TOP_HITS scoring matches for at least one of the features then it is deleted This step is necessary to removes some of the noise resulting from large gene families 2 2 2 Numbering of points Following this each point in the grid is given a unique identifying number from zero to one less than the total number of points These numbers are assigned to points in sorted hierarchical order first by one coordinate and then by the other Cells for which 7 gt 3 are not included 2 2 3 Enforcing symmetry The above preprocessing steps have ensured that symmetry has been enforced in the grid such that the match between features 2 and 7 has the same score as the match between features 7 and 2 Thus we only need to consider the half of the grid containing versus 7 in searching for blocks 2 2 4 Defining the neighborhood size
21. contigs which consist of a sequence of features A feature is typically one or more closely related protein coding genes at a particular locus but may be any entity to which 1t is possible to ascribe homology to other features Because FISH considers a contig to be merely an ordering of features it is in effect treating the distance between adjacent features as one unit 2 1 Step 1 From markers to features The names positions and transcriptional orientations when known of the markers are read from a set of map files one map file per contig Markers within each map file must be ordered according to their physical positions on the contig FISH assigns each marker a unique identification number from 1 to the total 9 10 CHAPTER 2 HOW IT WORKS number of markers in all the contigs which serves as a linear coordinate for subsequent calculations Markers cannot appear more than once on a contig i e each marker on a contig must be unique otherwise an error will result Individual homologies between markers are read from a set of match files There 1s at least one and no more than two such files for each pair of contigs Each match is associated with a score that reflects the strength of that match Larger scores indicate stronger matches When two matches between the same markers are present in two different match files i e contig 1 vs contig 2 and contig 2 vs contig 1 the score is taken to be either the maximum or the average of
22. contigs to be analyzed Each of those lines contains an integer followed by whitespace followed by an integer followed by whitespace followed by a valid filename path The two integers specify the contigs for the match file Note that there must be at least one comparison in which the integer of the first contig is less than the integer of the second contig For example if 1 versus 2 does not exist than 2 versus is ignored So if there is only one match file for a comparison between two contigs then that comparison must be written as 1 versus 2 4 1 2 Map files Each map file lists the names and transcriptional orientation if known of all the markers on one contig For an example see Section 5 Markers must be listed in positional order Each line contains a unique marker name followed by a tab character followed by the transcriptional orientation Marker names can be character string up to a maximum length of 100 that does not include whitespace or the amp character This value can be modified in the file fish cpp by changing the value of MAXBUFFER and recompiling Marker orientation is taken relative to the contig as a whole Thus if marker 1 is coded on the same strand as marker 2 then both markers should be assigned the same direction Orientation can take values 1 0 and 1 Zero is used for markers of unknown orientation 4 1 3 Match files Each match file lists all the homologies between markers in a
23. dvised of the possibility of such damages Because the program is licensed free of charge there is no warranty for the program to the extent permitted by applicable law Except when otherwise stated in writing the copyright holders and or other parties provide the programas is without warranty of any kind either expressed or implied including but not limited to the implied warranties of merchantability and fitness for a particular purpose The entire risk as to the quality and performance of the program is with you Should the program prove defective you assume the cost of all necessary servicing repair or correction 6 CHAPTER 1 GETTING STARTED In no event unless required by applicable law or agreed to in writing will any copyright holder or any other party who may modify and or redistribute the program as permitted above be liable to you for damages including any general special incidental or consequential damages arising out of the use or inability to use the program including but not limited to loss of data or data being rendered inaccurate or losses sustained by you or third parties or a failure of the program to operate with any other programs even 1f such holder or other party has been advised of the possibility of such damages 1 4 Acknowledgements Funding for FISH was provided by the USDA ARS Center for Agricultural Bioinformatics the Center for Computational Genomics at the Cornell Theory Center and by National Scien
24. en entirely by you rather the intent is to exercise the right to control the distribution of derivative or collective works based on the Program Aggregation of another work not based on the Program with the Program or with a work based on the Program on a volume of a storage or distribution medium does not bring the other work under the scope of this License You may modify your copy or copies of the Program or any portion of it thus forming a work based on the Program and copy and distribute such modifications or work under the terms of Section 2 above provided that you also meet the following conditions a You must cause the modified files to carry prominent notices stating that you changed the files and the date and nature of any change b You must cause any work that you distribute or publish that in whole or in part contains or is derived from the Program or any part thereof to be licensed as a whole at no charge to all third parties under the terms of this License These requirements apply to the modified work as a whole If identifiable sections of that work are not derived from the Program and can be reasonably considered independent and separate works in themselves then this License and 1ts terms do not apply to those sections when you distribute them as separate works But when you distribute the same sections as part of a whole which is a work based on the Program the distribution of the whole must be on the terms of this
25. ff none AVG_SCORE A Max Score none T T 0 05 real between O and 1 DETANDEMIZE off on none 14 3 1 ALGORITHMIC PARAMETERS 13 3 1 Algorithmic parameters A number of the adjustable parameters control the way in which FISH finds blocks Altering these parameters can substantially influence the results MIN BLOCK SIZE The minimum number of points needed to define a homologous block TOP HITS Matches that are not in among the TOP HITS scoring matches for either component feature will be discarded MAX_DIST The maximum number of intervening markers allowed between markers in t he same detandemized feature T The probability of having one or more neighbors within a distance less than or equal to than dy under the assumption that each cell contains a point with probability h Values of T closer to zero will result in blocks with more closely spaced points This is roughly analogous to the gap parameter in local sequence alignment MIN_SCORE Matches with scores less than MIN_SCORE will not be considered by FISH despite being listed in a match file AVG_SCORE This option enforces symmetry between two matches e g 1 versus 2 and 2 versus 1 by taking an average of the scores If this averge is less than the MIN_SCORE the match will be discarded The default behavior is to choose the larger of the two scores DETANDEMIZE This allows the user to turn detandemization off 3 2 Input and output parameters The remaining parameters co
26. ile name Processing contigs FISH displays the values of the two relevant parameters for the contig processing stage analysis MIN SCORE and MAX DIST Once detandemization 1s complete a table is written that displays the number of markers and features within each contig Detailed information on which markers compose which features is written to a file if the PRINT_CONTIGS parameter has been selected Processing matches FISH displays the values of the relevant parameters of the match processing stage MIN_SCORE and TOP_HITS Note that the current and previous value for for MIN_SCORE must always agree Once the calculations are complete FISH displays the number of points and the number of cells for each pair of contigs in the analysis In addition the coordinates of the points in the detandemized dataset are written to a file if PRINT_GRIDS has been selected 20 CHAPTER 4 FILE FORMATS Processing blocks FISH first displays the key adjustable parameter of the block finding stage T Following this is a series of numbers which depend on both the dataset and the adjustable parameters e N the total number of points in the grid e F the total number of features in the dataset e the total number of cells in the grid calculated as F F 1 2 e h the proportion of cells in the grid that contain a point e dr the neighborhood size within which there is only a T probability of encountering another point if points in the grid are di
27. inear order As described more fully below FISH evaluates the probability of observing each block under a null model in which matches are distributed uniformly at random among features Users of FISH should cite the following paper in which more detail regarding the method can be found e Calabrese PP Chakravarty S Vision TJ 2003 Fast identification and statistical evaluation of segmental homologies in comparative maps Bioinformatics 19 174 180 2 CHAPTER 1 GETTING STARTED 1 2 Contact information Please send questions feedback and bug reports to tjv bio unc edu The FISH website provides an online form where you may submit your email address should you wish to receive notification about updates to this software 1 3 Legal information FISH and its associated documentation are copyright 2003 University of North Carolina at Chapel Hill FISH is distributed in the hope that 1t will be useful but without any warranty whatsoever even the implied warranty of merchantability or fitness for a particular purpose Use of this software implies acceptance of the terms of the following license As used herein the term Program below refers to the source code and the derivatives supplied by the authors and a work based on the Program means either the Program or any derivative work under copyright law that is to say a work containing the Program or a portion of it either verbatim or with modifications and or translated
28. is License 7 SEVERABILITY If any portion of this license or any portion of any section of this license is held invalid or unenforceable under any particular circumstance the balance of the section is intended to apply and the section as a whole is intended to apply in other circumstances 8 NO WARRANTY Because the program is licensed free of charge there is no warranty for the program to the extent permitted by applicable law Except when otherwise stated in writing the copyright holders and or other parties provide the program as is without warranty of any kind either expressed or implied including but not limited to the implied warranties of merchantability and fitness for a particular purpose The entire risk as to the quality and performance of the program is with you Should the program prove defective you assume the cost of all necessary servicing repair or correction In no event unless required by applicable law or agreed to in writing will any copyright holder or any other party who may modify and or redistribute the program as permitted above be liable to you for damages including any general special incidental or consequential damages arising out of the use or inability to use the program including but not limited to loss of data or data being rendered inaccurate or losses sustained by you or third parties or a failure of the program to operate with any other programs even if such holder or other party has been a
29. med data the names would read e g datamap1 txt on a Unix system and would read datal map1 txt on a Windows system maps 23 24 CHAPTER 5 AN EXAMPLE data mapl txt data map2 txt data map3 txt data map4 txt data map5 txt matches 1 data matchlvl txt 1 data match2v1 txt 1 data match3vl txt 1 data match4vl txt data match5vl txt data matchlv2 txt data match2v2 txt data match3v2 txt data match4v2 txt data match5v2 txt data matchlv3 txt data match2v3 txt data match3v3 txt data match4v3 txt ta match5v3 txt data matchlv4 txt data match2v4 txt data match3v4 txt data match4v4 txt data match5v4 txt data matchlv5 txt data match2v5 txt data match3v5 txt data match4v5 txt data match5v5 txt Oe NH oP GA NP OG TD OB WN O1 O1 O1 O1 OD VG ds ds ds a GO GO GA UY YN NN NN N DN EF Q HU 5 2 A map input file Shown are the first ten lines from the chromosome 1 map file map txt The first column is the marker in this case gene name the second column is the 5 3 A MATCH INPUT FILE 25 transcriptional orientation At1g01010 1 At1g01020 At1g01030 A At1g01040 1 At1g01050 1 At1g01060 At1g01070 1 At1g01080 At1g01090 At1g01100 5 3 A match input file Shown is a sample of ten lines from the chromosome 1 vs chromosome 1 match file match 1v1 txt
30. ntrol the input to and output from FISH QUIET MODE If selected FISH will suppress the default screen output see 4 2 1 This switch takes no arguments TIMING If selected FISH will report the number of seconds elapsed between various execution time points This switch takes no arguments 16 CHAPTER 3 PARAMETERS CONTROL FILE Specifies the name of the file containing the instructions for FISH to read particular contig and match files 4 1 1 If not specified at the command line FISH will search for a file named control txt and use that 1f available PRINT_CONTIGS Specifies the name of the file to which FISH writes contig output see 4 2 2 Unless selected no file will be written PRINT_GRIDS Specifies the name of the file to which FISH writes grid output see 4 2 3 Unless selected no file will be written PRINT BLOCKS Specifies the name of the file to which FISH writes block output in human friendly format see 4 2 4 Unless selected no file will be written PRINT_B_SIMPLE Specifies the name of the file to which FISH writes an alternative format for the block output that is more computer friendly see 4 2 4 Unless selected no file will be written BLOCK_PROB This specifies a value for the confidence level of a block The confidence level is calculated as the expected number of blocks with n points divided by the observed number of blocks with n points If the observed number does not sufficiently exceed the expected
31. other pertinent obligations then as a consequence you may not distribute the Program at all For example if a patent license would not permit royalty free redistribution of the Program by all those who receive copies directly or indirectly through you then the only way you could satisfy both it and this License would be to refrain entirely from distribution of the Program It is not the purpose of this section to induce you to infringe any patents or other property right claims or to contest validity of any such claims this section has the sole purpose of protecting the integrity of the free software distribution system which is implemented by public license practices Many people have made generous contributions to the wide range of software distributed through 1 3 LEGAL INFORMATION 5 that system in reliance on consistent application of that system it is up to the author donor to decide if he or she is willing to distribute software through any other system and a licensee cannot impose that choice 6 LICENSE CONVEYS TO SUBSEQUENT RECIPIENTS Each time you redistribute the Program or any work based on the Program the recipient automatically receives a license from the original licensor to copy distribute or modify the Program subject to these terms and conditions You may not impose any further restrictions on the recipients exercise of the rights granted herein You are not responsible for enforcing compliance by third parties to th
32. ough third parties are not compelled to copy the source along with the object code 5 LICENSE RESTRICTIONS a You may not copy modify sublicense or distribute the Program except as expressly provided under this License Any attempt otherwise to copy modify sublicense or distribute the Program is void and will automatically terminate your rights under this License However parties who have received copies or rights from you under this License will not have their licenses terminated so long as such parties remain in full compliance b You are not required to accept this License since you have not signed it However nothing else grants you permission to modify or distribute the Program or its derivative works These actions are prohibited by law if you do not accept this License Therefore by modifying or distributing the Program or any work based on the Program you indicate your acceptance of this License to do so and all its terms and conditions for copying distributing or modifying the Program or works based on it c If as a consequence of a court judgment or allegation of patent infringement or for any other reason not limited to patent issues conditions are imposed on you whether by court order agreement or otherwise that contradict the conditions of this License they do not excuse you from the conditions of this License If you cannot distribute so as to satisfy simultaneously your obligations under this License and any
33. rhood de is the distance of the cell from the point under consideration and w is the weight The weight 1 1f the point is the first in the block w 30 25 if an inversion is implied by the extension 2 3 0 75 otherwise When multiple points have equal rank the point is chosen that has the smallest value of X X Y Y This favors points close to the diagonal 2 3 2 Overlapping blocks FISH processes blocks in such a way as to ensure that only non overlapping blocks are obtained If a point could be included in multiple blocks one of the 2 3 STEP 3 FROM GRID TO BLOCKS blocks with highest score is chosen The point is then made unavailable for inclusion in other blocks 13 Chapter 3 Parameters A number of parameters may be adjusted at the command line when FISH is invoked They may also be reviewed from the command line by invoking FISH with the help switch fish h The adjustable parameters are listed here and described in more detail below parameter switch default acceptable values PRINT_BLOCKS b off valid file name PRINT B SIMPLE B off valid file name MIN BLOCK SIZE m 3 integer greater than 1 PRINT_CONTIGS C off valid filename CONTROL FILE f Control DI valid filename PRINT_GRIDS g off valid filename TOP_HITS H 5 integer greater than O MAX_DIST D 10 integer greater than O BLOCK_PROB p 0 001 real between 0 and 1 QUIET MODE q off none MIN_SCORE S 200 real TIMING t o
34. stributed uniformly at random see Equation 2 1 e MIN BLOCK SIZE the number of points in the smallest block to be reported Once the blocks have been identified FISH will also display the total number of blocks The length distribution of the blocks is displayed as a vertical histogram To the right of each bar is listed the number of blocks of length L as measured by the number of points If the number of blocks is greater than 50 the true amount is not represented by characters and the block frequency is preceded by To the left of the histogram is shown the corresponding p value for observing a block of length L see Section 4 2 4 and the expected number under the null model If the proportion of expected to observed blocks of length L is greater than BLOCK_PROB then the frequency of observed blocks is followed by an exclamation mark This indicates that blocks of this length have not been observed sufficiently more frequently than expected and so the individual blocks may not be biologically meaningful 4 2 2 Contig file If the PRINT_CONTIGS option is selected FISH will output a contig file with the specified name See Section 5 for an example The contig file allows 4 2 OUTPUT 21 translation between feature numbers and marker names The top of the contig file displays the time at which the process was initiated This is followed by the parameter list which includes the name of the control file and the v
35. the two according to the user s choice refer 3 On the basis of the map positions and matches FISH performs detandemization in which multiple markers may be collapsed into single features Two parameters control the detandemization process MIN SCORE and MAX DIST Markers with coordinates that are separated by MIN DIST or less will be collapsed into a single feature if they are homologous to each other or to a third marker with a score at least as great as MIN SCORE To calculate the relative order of features in the detandemized contig each feature inherits the position of the marker having the lowest valued coordinate within the set of markers comprising the feature The coordinates are then reassigned for features in all contigs from zero to one less than the number of features in the whole dataset When transcriptional orientations are known the feature inherits the majority orientation either 1 or 1 of the markers that comprise it For example if three out of the five markers that comprise a feature have a transcriptional orientation of 1 then the feature inherits that orientation If there is no majority then it is assigned a value of zero for unknown The score of a match between two features when one or both of the features represents multiple markers is taken depending on the runtime parameter chosen to be either the highest score or the average score for any of the pairs of component markers one from ea
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