T rnsformer - 2
Contents
1. alete Heterodimer Add Format Records Figure 18a Select the cell where you want to add a new allele alete Heterodimer Add Format Records a O EEE L Locus m EnzvmejPrimer ADH Ze Species Population 1 Sheet Figure 18b From the custom toolbar select Add and then Allele Hebe Heterodimer Add Format Records ADH Gntp Insert Allele This new allele will he assigned to locus mm mmm CANCEL Figure 18c In the dialog box that appears the new allele can be assigned either to any of the two flanking loci only red in the example or to any other existing locus by selecting NONE TEM eee ee ee s MANUAL 24 Juli Caujap Castells and Mario Baccarani Rosas ilete A Heterodimer Add Format Records E EEEEE BEBE Bw BX ADH ken SETTER A B B EE EE EE 0H 1 ADH 2 Figure 18d If we pressed the red button in Fig 18c a new allele would be assigned to the red locus ADH 1 in the selected position alete Heterodimer Add Format Records f Pattern Inter Insert Allele X Allele coo NN OK CANCEL Basic Allele Colors WARNING If you select one ofthe colors above then the new allele will be assigned to one ofthe existing loci Degraded Colors If you wantto make a mark and you dont want this mark to be interpreted by the program use any ofthe colors above Figure 18 e If we pres2. Enzyme primer header Genotype area Drawing matrix SB Es Sample codes Population codes Species codes Sample numbers Figure 2 Detail of the drawing matrix of 7ransformer 2 The first four columns of the area coloured in light grey correspond to the sample numbers N the species names Species the population codes Code and the sample codes Sample The next columns in grey show the enzyme primer header for the enzyme MDH which in this case has two loci with two and five alleles respectively and the genotype area The white area below the enzyme primer header is the drawing matrix where the user can draw the interpretations of gels following the indications in the manual Only a part of the drawing matrix for MDH is shown TEM eee ee ee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas 3 The drawing matrix for a given enzyme must have at least 11 columns so that if you reach this minimum width 7ransformer 2 will not allow you to delete columns see section 1 2 4 and an error message like the one below will appear Delete Column KS AN You are not allowed to delete one more column See section 1 1 2 in the manual 4 For the sake of uniformity 7ransformer 2 assigns a predefined colour to all the alleles belonging to a given locus All the alleles of the first locus within an enzyme primer will be red those at the second black those at the third blue and so on until the
3. Juan Francisco Rodr guez Izzat Sabbagh and Gonzalo Piernavieja at the Departamento de Ingenier a del Software of the ITC are acknowledged for their receptiveness to the idea of collaborating in the development of this program and for their continued support and interest TEM eee ee ee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas PREFACE The history behind 7ransformer 2 is in short another one of chance and necessity Necessity came along with the growing bulk of data analyses related to the population genetic projects under way at the Jard n Bot nico Canario Viera y Clavijo JBCVC that triggered the creation of a 7ransformer 1 Caujap Castells 2001 That first version proved to be suitable enough to bypass a number of burdensome and error prone aspects of molecular population genetic data analysis though it was still too tangled to be released without shame Therefore 7ransformer 1 was only operated by Juli Caujap Castells in his personal computer Available time was in very short supply since the creation of 7ransformer 1 and this alone would have provided an excellent excuse not to pursue a better version after all that first program already analysed our molecular population genetic data much faster than usual Perhaps the chance to develop a 7ransformer 2 wouldn t have turned up had it not been by Eugenio Reyes an educator at the JBCVC who was aware of the Transformer project This person put Ju
4. Matt WCH 2 H ALLELES E 1 z i 1 E E r L LJ LJ HH 8 ZE SEXEHE EZZZMZxzume Figure 12b Defining the basic features of the new Enzyme primer In this case allozymes the enzyme MDH has two loci MDH 1 and MDH 2 with 3 and 5 alleles respectively If you are drawing allozymes write a the code of the enzyme b the number of loci and c the number of alleles for each locus TEM eee eee s MANUAL 19 Juli Caujap Castells and Mario Baccarani Rosas If you are drawing microsatellite profiles just a introduce the primer code in the corresponding cell b put a 1 in the box number of loci and c introduce the number of alleles you re about to draw 3 press OK Transformer 2 will then ask you if everything is correct If you confirm the number of alleles that you have selected for each locus in a given enzyme primer will appear automatically below the enzyme primer header with their corresponding colour and letter codes Figure 13 illustrates the default conformation of the drawing matrix for the selection made in Fig 8 Since we selected 2 loci with 3 and 5 alleles respectively there will be 3 red bands and 5 black ones Move Left Insert WY Delete 4k Heterodimer Add Format Records JO Pattern Interpre E16 M 6 d ES oficinais 2 amp officinalis POP1 3 A officinalis POP 1 2 3 4 officinalis POP1 4 5 officinalis
5. Castells and Mario Baccarani Rosas 1 2 8 Drawing phantom bands Those working with allozymes are used to come across bands whose interpretation is thorny because they cannot be assigned safely to any locus In most cases it is convenient to store these so called phantom bands Ar s and Shields 1983 as qualitative information for eventual consideration in the future At the JBCVC students that use molecular population techniques are always requested to draw the phantom bands if any to purport a more realistic version of the gel that can set the stage to alternative interpretations This utility can also be used to represent heterodimers Transformer 2 offers two degraded versions of the colour codes assigned to each locus to represent these phantom bands see example in Figure 21 Insert Allele E Allele Color OK CANCEL Basic Allele Colors WARMING If vau select ane ofthe colors above then the new allele willbe assigned to one af the existing loci Degraded Colors If vau want Io make a mark and you dontwantthis mark to be interpreted by the program use any ofthe colors above Figure 21 Palette of possible colours for phantom bands below the heading Degraded colors for each of the 10 corresponding loci colours Notice that each locus colour has two different associated tones that can be used to represent phantom bands See sections 1 1 5 and 1 2 8 for details on the use of these bands 1 2 8 1 I
6. You first have to request 7ransformer 2 to analyse the enzyme primer patterns one by one This process allows you to check any possible error more easily than if all the patterns were analysed at once To analyse the patterns just TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas a Select Pattern interpretation in the toolbar menu Figure 24 fo Pattern Interpretation Figure 24 b Fill in the box that will appear with the name of the enzyme primer whose pattern you want to analyse see Figure 25 the Enzyme primer to interpret must be in the active sheet 4 Move Left 4 Insert HF Delete 4 Heterodimer Add Format Records L n c N Species Code Sample B RH 1 ovem POP1 1 B E 2 R officinis POP1 2 B D 3 mom POP1 3 B E LP 4 Goes POP1 4 8 E LP 5 moe POP1 5 B E N 6 Goes POP1 6 B E N Y R officiais POP1 7 B RH E 8 moe POPI1 e B E N 9 mof POP 3 H B N m none an LJ m I a 0 m 11 Enzyme Primer Interpretation X 12 13 Enzyme Primer MDH OK CANCEL 14 15 16 Gov POP2 16 17 2 officiais POP2 17 B 18 Wm S POP2 18 HA E Figure 25 Selecting an enzyme primer for interpretation C Press OK and 7ransformer 2 will genotype that enzyme primer Figure 26 4 amp Move Left t Insert HP Delete Y Heterodimer Add Format Records D BF19 A DCES Eb E 3 Gntp ES A C 5 N Species Co
7. specifications in section 1 2 2 b The second line and the subsequent ones contain the data DO NOT start writing your data in the first line 1 N Species Code MDH 1 MDH 2 IDH 1 1a officinalis POPL AC SS BB 2R officinalis POPL AC CC BB 38 officinalis POPL AA BD BB dn officinalis POPL AA CE BB EI 5 amp officinalis POPL AA BD BC GR officinalis POPL AA BD BE E TR officinalis POPL AA AE BD 9 os officinalis POPL AA CE BE OR officinalis POPL cc BD BB 108 officinalis POPL AA BD BB 11 officinalis POP AA BD BB l2 officinalis POP2 AA BD BB 13 R officinalis POP2 AC CE BE l4 R officinalis POP2 AA ae BB 154 officinalis POP2 AA CE BB L ps officinalis POP2 AA ED BE 17 R officinalis POP2 AA AE BC 18a officinalis POP2 AB ae BB A mln A a c Ma D ux m m E A A gd mam Figure 30 Detail of the format of a genotype sheet for entry in 7ransformer 2 This image corresponds to the first individuals and loci in the attached file transf gntp ex oe eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas Section 2 Configuring the data The starting point of the data configuration capabilities of Transformer 2 is the matrix of genotypes either obtained through the drawing sheet or implemented ad foc To configure your data for analysis 1 Click on the button Configure data from the toolbar of Transformer 2 see below Configure Data sole mii B 2 The big dialog box that will appe
8. with the program before you input your own real data TEM eee eee s MANUAL 38 Juli Caujap Castells and Mario Baccarani Rosas EE MANUAL 59
9. A A Eo E E Figure 19c Transformer automatically inserts the new locus with its corresponding colour The new locus can be inserted at any position in the drawing matrix corresponding to a given enzyme primer If you place it in the middle of two pre existing loci then 7ransformer 2 will automatically refurbish the colour codes of the loci at the right of the newly inserted one so that they fit the new conformation The program will also insert a new column in the genotype area Figure 20 illustrates the effects of the insertion of a new locus between two pre existing ones labelled in red and black Transformer 2 will re draw the alleles in black so that they now will belong to the new locus 3 which should be blue according to the colour code while those for locus 1 will remain untouched because they are at the lefthand side of the locus and they are not affected by the appearance of the new locus 1 sheet i cus L SE Be Ence Primer ESTE ls ETE 25 A L H a L A nn oa on A L L A L L a L Figure 20 Insertion of a new locus between two pre existing ones Note that after inserting the new locus if there were any loci at the right side of it the colours of all their alleles change automatically according to the colour codes in Fig 3b In the case of this figure the alleles in the locus that was previously black changed to blue after the insertion TEM eee eee s MANUAL 27 Juli Caujap
10. Delete 4 amp Heterodimer Add E6 bi E E HUDHIDDAAD DAHA 13 18 18 18 18 S Al LEE Soe cd eo A oh a o GG M m C 4 L bM e 9 2 Figure 10 Detail of the drawing sheet after pressing OK with the selection made in Fig 9b ve ebe MANUAL Juli Caujap Castells and Mario Baccarani Rosas C Insert the remaining populations of your project in the Transformer 2 drawing sheet by selecting Species in the Add button from the toolbar as many times as needed see Figure 11a b and c a b d Za dimindi WU P own d hnil on f S gt Bi A e Sang AE E Kiva nl 4 heel Y Parle Hd run bas bl e Puma Brin E04 dan im redii m LET 4 d dave Left Y inser J Delete etacdper add a 5 SEREHE Code m INL MW Mena POF Lo NM Mena POF Screw POF1 11m Aa POF 1 m INL MW Mena POF en POF1 m roa Zen POP m IN Ne Mena POF INN Mena POF Screw POF m INL Ne Figure 11 Adding new populations to the 7ransformer 2 drawing sheet 1 2 2 Choosing population codes For a population code you can use any string of characters including numbers and signs The only restriction is to choose codes without empty spaces whose symbols if any do not conflict with the entry formats of any of the programs that 7ransformer 2 generates files for see section 2 Some examples of two population codes that 7ransforme
11. Imagine we want to check the effect of removing sequentially single populations in the value of some parameter that can be calculated using 7ransformer 2 The selections to make would be like in Figure 40 Selecting Populations OS 7 PopGene GeneStat Biosys NTS 5 pe GenePop Bottleneck Frob Lass EXIT GROUP GROUPS GROUPS GROWP4 GROUPS GROUPE GROUP POP1 POP POPJA FOP3B POP4 POPS POPE SEISPISTSHSPISIIS L MSHSHSHSHSISI H HSPHISHSHISHSI naa aaa HHH Alas H HHH HSHSI GR Figure 40 4 Press the button corresponding to the transformation s you want to perform for that conformation of your data give adequate names to the files that will be generated and press save see section 3 After completing this sequence the files are ready to be run in the specific programs for which they were formatted TEM eee eee s MANUAL 45 Juli Caujap Castells and Mario Baccarani Rosas Section 3 Processing the data For each configuration of populations 7ransformer 2 generates automatically the necessary files to run six population genetic analysis programs and calculates all the parameters related to the probabilities of allelic loss 3 1 THE BIOSYS FORMAT BIOSYS Swofford and Selander 1989 is a Fortran IV computer program that can be used to calculate the values of most population genetic polymorphism indicators test for Hardy Weinberg equilibrium compute statistics perform heterogeneity chi squ
12. adequate names to the files that will be generated and press save see section 3 After completing this sequence the files are ready to be run in the specific programs for which they were formatted see section 3 If you have used the file transf gntp xls to follow this explanation try some of the options 2 2 3 Including populations in more than one group In some cases the population geneticist might be interested in testing how the values of different parameters change depending on which populations are included removed from a given group For TEM eee eee s MANUAL 43 Juli Caujap Castells and Mario Baccarani Rosas these and other similar cases 7ransformer 2 allows the user to include any population in different groups in the following way 1 Select the total number of groups to analyse In the example of Figure 38 imagine we want to define seven groups with the data in the file transf gntp xls ed Seck ee POP1 POP2 POP3M POP3B POP4 POPS POPE B3 E EE 35 EH N M amp HHO M n gu gu EE Figure 38 2 Label the groups with a proper name In Figure 39 we just choose the labels GROUP1 to GROUP7 Selecting Populations aoe ee AA ees Aa ee AE ia ROWIP 4 GROUP GROUPS GROUPS GROUPS GROUPB GROUP E E m Ed B 8 EM ME 8 HM M ERR M Figure 39 TEM eee eee ss MANUAL Juli Caujap Castells and Mario Baccarani Rosas 3 Select the populations to be included in each group
13. do this 1 Press on the button Ntsys in the dialog box 3 Give a proper name to each of the three files Transformer 2 reminds you what Ntsys file you are about to save The first Ntsys file it creates is the allele frequency file the input file for Ntsys that appears in the dialog box with the default name ntsys frequencies Figure 41 just re name the file as you wish Creating NTSYS Frequencies File Escritorio Favoritos 1 tsys Frequencies AAA Guardar como tipo ursvs Files nts E Cancelar Figure 41 oe eee eee s MANUAL 20 Juli Caujap Castells and Mario Baccarani Rosas Once you save this input file the second Ntsys file that Transformer 2 will create for the configuration of populations you defined is the loci array file that contains the number of alleles per locus This file appears in the dialog box with the default name ntsys alleles Figure 42 just re name it as you wish Creating NTSYS Alleles File 2 x Guardar en Corel User Files SCH Mis im genes Historial 9 my archives My eBooks EI all nts a freg mts Mis documentos EI ntsys frequencies nts aj sample nts Nombre de archivo TEER Es Guardar como tipo DES Files nts Cancelar Figure 42 Finally the third Ntsys file that 7ransformer 2 creates for the configuration of data you defined is the N array file that contains the corresponding sample sizes This file appears i
14. example files transf draw xls and transf gntp xls are attached without changes and iii it is adequately cited in all papers and communications Transformer 2 is provided as is without any kind of warranty In no case will the authors or their supporting institutions be liable for any trouble resulting from the use of this software or of its accompanying documentation Suggestions criticisms and bug reports on 7ransformer 2 are very much welcome Address them to julicaujapeOgrancanaria com orto mbaccaraniOwanadoo es TEM eee ee ee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas PLEASE CITE 7RANSFORMER 2 IF YOU USE IT No one is obliged to download ransformer 2 Therefore if you use this program please cite it This is how Caujap Castells J Baccarani Rosas M 2004 7ransformer 2 a program for the analysis of molecular population genetic data Jard n Botanico Canario Viera y Clavijo and Instituto Tecnol gico de Canarias Las Palmas de Gran Canaria Spain The support we receive through your citations is also very important in order to facilitate our seeking the necessary means to improve the program further FUTURE RELEASES Transformer 2 is already being improved to include a much wider range of possibilities We hope that a new version that will allow RAPD and AFLP data analysis will be ready before spring 2005 In a longer term we plan to have a 7ransformer that performs most of the calc
15. last one of the pre existing ones see Fig 23d e Write in the dialog box the number of individuals you want to add Figure 23d After completing this process the corresponding number of cells will appear after the last of the pre existing individuals in the selected population see Figure 23e Notice that for the newly added TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas individuals 3 in the example the cells in the column Sample are blank so that you can insert the eventual code of the new samples their species and population codes will remain the same as for the other individuals from that population Also notice that the sample codes for the pre existing individuals will remain the same but their sample number N will have varied according to the number of inserted samples see Figure 23e and note the changes in N in POP2 SS SES EKEN E SIETE EIEI E EE 1 Lie T 2 2257 xXagiNSal ar 25 0 nude adsl i QUSE x FI FIFI FI FI FI FI FI ee eee rte Tr Y TOY Y YT Y TIT AECLEEEEEEEEE EE ERES did ae Pelele El Hj 1 1 SCH ae enun s NEN a ae DT l Figure 23 Inserting new individuals in a pre existing population 1 2 10 Get the genotypes from the drawing When you complete the drawings for all the loci and individuals you have included in your project you are ready to obtain the genotypes
16. you pressed Create Chart and c the value of representativity A if you pressed Create Chart 3 8 TIPS ON PROCESSING THE DATA After obtaining the datafiles and analyses for a given configuration of populations you can go back to the original matrix and define another configuration of interest This way you can get all files you want to analyse for as many configurations you may be interested in before running the corresponding programs or carrying out calculations TEM eee eee s MANUAL 22 Juli Caujap Castells and Mario Baccarani Rosas LITERATURE CITED Ar s P Shields CR 1983 Cole crops Brassica oleracea L Isozymes in Plant Genetics and Breeding Part B Elsevier Publishers Amsterdam Bengtsson BO Weibull P Ghatnekar L 1995 The loss of alleles by sampling a study of the common outbreeding grass Festuca ovina over three geographical scales Hereditas 122 221 238 Caujap Castells J 2001 Transformer 1 0 Un programa de formateado de datos genotipicos individuales para implementaci n en programas de an lisis molecular en gen tica de poblaciones Caujap Castells J Pedrola Monfort J 2004 A sampling design for the ex situ genetic conservation of the Ibero Moroccan endangered endemic Androcymbium gramineum implications for the assessment of a conservation strategy from a survey of genetic diversity for neutral markers Conservation Genetics 5 131 144 Caujap Castells J 2004 Manual para el muestr
17. 0 12 Average allele frequencies Figure 46 Linear regression of the average allele frequencies and the log Lo black circles continuous line and log Le black circles discontinuous line for all the alleles that fulfill the Viera y Clavijo conditions of rarity in the example file transf gntp xls At the header of the chart the program indicates the number of alleles that are included in the representation and calculates the representativity value as described in the text EE MANUAL Juli Caujap Castells and Mario Baccarani Rosas Transformer 2 also calculates the value for the representativity R of sampling only one population of that group relative to the total sample of rare alleles by dividing the slope of the observed regression line based on the values of Lo by the slope of the expected regression line based on the values of Le Bengtsson et al 1995 3 7 2 Obtaining the probabilities of allelic loss 1 Press on the button Prob Loss in the dialog box 2 Select the option that you want to use see section 3 7 1 3 Press OK 4 Give a proper name to the Excel output file The resulting Excel file will contain a a Table of allele frequencies for each of the groups selected b a Table with the values of Z for the alleles that fulfilled the conditions of the calculation option that you selected below the previous one d the graph with the linear regressions commented in section 2 3 7 1 if
18. Juli Caujap Castells and Mario Baccarani Rosas a Write the number of groups you wish to establish in the corresponding cell and then press Return In the example in the file transf gntp ex with 7 populations imagine that populations POP1 POP3A and POP3B belong to a Species 1 and the remaining populations to a Species 2 If we wanted to compare these two species we would write a 2 in the cell labelled Groups Figure 35 POP ll POPE Figure 35 b Re name the matrix column headers in the box to label the groups you want to define In our example we choose the labels SP1 and SP2 Figure 36 Selecting Populations E E weno men ne p Sassel SO 522 Figure 36 TEM eee eee ss MANUAL Juli Caujap Castells and Mario Baccarani Rosas C For each of the groups tick the boxes that correspond to the populations that they must contain In the example group SP1 consists of the populations POP1 POP3A and POP3B while group SP2 consists of POP2 POP4 POP5 and POP6 Figure 37 illustrates the aspect that the configuration matrix would have in this case Selecting Populations KE cu 2 PopGene Genestat Biosys NTSys pe GenePop Bottleneck Prob Loss EXIT P2 SP cen POP POP POPA POP3B POP4 POPS POP6 EL Heel He ell H SHE Figure 37 d Press the button corresponding to the transformation s you want to perform for that conformation of your data give
19. LIEnsTormer Castells 1 Caujap amp Mario Baccarani Rosas Jul jep 9139436 uonejndod Iejnosjow jo sisAjeue ou 104 WeIBold y Pi CN A CeL EA LOLA A ro PI II IO je je p se IO 245 22 l2 c2 c2 c2 c2 2 2 p oil 25 22x 2 5 2z c2 uz A A L a CA O sz o vu s PERE GE SAE SIESTA a rimo wo wi 8 DIRGIE SICOrnt iE GE J3E d itd td td tzd tz t diro nds cgi da D 7 7 D DEED BEDEELEGT EEGEN EE EE DEE EE EEN ach araara cazia TE AER EH EEN iilrzibiialiuliilio BH En E EE mmm mmm mm mm mm mmm EH mm mm mum mmm mmm mmm mmm mmm mmm mmm mm LIII NEL Nl ee lr IE E m m m m mm Um mmm m m Gm Um Hum m m ERE mmm mmm mm mmm mmm mmm mmm mmm Se m E m n MN HON O IO DO O DOERR SOROR DOO DONO DOG NONO DONO GO GNO DONE IER DN mmm OBI EI mm mmm mmm mmm mmm mmm mmm mmm mm mm E mmm mmm SONS E mmm m BERR RE mmm mmm mmm hme NONO D mmm m m m m m m u EN E mmm mmm mm mmmm A Dm mm m m HEE HEHE mm BEDE NENNEN E E HEE mmm mm mmm eee vn D DER Um m mmm m um m L BE EERE mmm E mm mmm mmm mmm mmm mm mmm mmm mmm mm mm mm L8 mmm mm um mmm mmm mm mmm mmm mmm mmm mmm NE DEN HEHEHEHE HE mmm mmm mmm mmm mmm mm mmm mmm mum EE m Juli Caujap Castells and Mario Baccarani Rosas ETE TARA TA User s manual by Juli Caujap Castells and Mario Baccarani Rosas Laboratorio de Biodiversidad Molecular Jardin Botanico Canario Viera y Clavijo Ap de Correos 14 de Tafira Alta 35017 Las Palmas de Gran Canaria Canary Isla
20. NTSYS pc Numerical Taxonomy and Multivariate Analysis version 2 02j Applied Biostatistics Inc Schonewald Cox CM Chambers SM MacBride B Thomas WL editors 1983 Genetics and conservation a reference for managing Wild animal and plant populations Benjamin Cummings Menlo Park CA Swofford DL Selander RB 1989 BIOSYS 1 a computer program for the analysis of allelic variation in genetics University of Illinois Urbana Ill Yeh FC Yang RC Boyle TBJ Ye ZH Mao JX 1997 PopGene the user friendly shareware for population genetic analysis Molecular Biology and Biotechnology Centre University of Alberta Canada EE MANUAL gt Juli Caujap Castells and Mario Baccarani Rosas APPENDIX THE TWO EXAMPLE FILES Transformer 2 comes with two interrelated example files for allozyme data One of them is an example of drawn interpretations transf draw xls and the other one is a genotype file transf gntp xls that corresponds to all loci that appear in the drawn interpretations you can obtain it by interpreting the patterns and then pressing Genotype file as described in this manual Both files consist of data for 12 allozymes 31 loci in 116 individuals that represent seven populations of the fictitious species R officinalis The drawings of these enzymes are distributed in two sheets within the same Excel file Sheet 1 contains the patterns for MDH IDH GOT PGM 6 PGD EST ME and PGI while sheet 2 contains the pat
21. POP1 5 6 7 8 6 A officinalis POP1 7 AR officinalis POP1 8 amp officinalis POP1 9 amp officinalis POP1 9 10 amp officinalis POP1 10 110 amp officinalis POP2 pl 12 amp officinalis POP2 12 13 amp officinalis POP2 13 14 amp officinatis POP2 14 15 amp officinalis POP2 15 16 A officinalis POP2 16 21 17 A oficinales POP2 17 18 amp oficinais Figure 13 The default conformation of the drawing matrix for the selection made in Fig 8 Also at the right of the drawing matrix for each enzyme primer there will be as many columns as loci you have defined each of them correspondingly coded and coloured These columns two in the example will remain empty until you decide to interpret your patterns see section 1 2 10 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas 1 2 4 Place the alleles in their correct positions As you can notice in Figure 13 the separation among alleles and loci is assigned automatically by 7ransformer 2 and it will probably not correspond to their real separation on the gels Thus a first thing you want to do is to adapt the relative positions of the alleles to reflect their positions in the gel Do it one allele at a time starting with the one at the far right as follows a Select the corresponding coloured cell in the enzyme primer header b Click on move left or move right in the bar chart menu until you have place
22. ar see Figure 31 contains the default options This box can already be used for data analysis and transformation see section 2 1 but it also constitutes the basis to implement the different configurations that you may want to give to your data see section 2 2 2 1 THE DEFAULT CONFIGURATION The dialog box that first appears when you click on the option Configure data contains the configuration of data that Transformer 2 would analyse by default see Figure 31 Selecting Populations Groups 7 PopGene GeneStat Biosys NTSys pe GenePop Bottleneck Prob Loss EXIT POP1 POP2 POP3A POP3B POP4 POPS POP6 3 WD a e 0 D a a o K e Lg M NM EM N Bu EN NI LEM M MEM 8 o uv e L L Figure 31 Default population configuration for the seven populations in the file transf ex xls ve ee eee ees MANUAL 37 Juli Caujap Castells and Mario Baccarani Rosas This box consists of three parts 1 The number of groups with the default value that corresponds to the number of different populations that 7ransformer 2 has detected in the table of genotypes 7 in the file transf draw xls 2 A series of buttons with the name of the options that you can invoke For any combination of populations 7ransformer 2 can give you the file formats needed to run your data in the following six programs Biosys see section 3 1 Bottleneck see section 3 2 G
23. are analysis calculate a variety of genetic distance coefficients construct phenograms and estimate phylogenies through the distance Wagner procedure 3 1 1 Obtaining the Biosys format 1 Press on the button Biosys in the dialog box 2 Give a proper name to the corresponding data file 4 Press OK 5 Your data are ready to run in Biosys By default the ASCII file that 7ransformer 2 creates for Biosys contains the following command lines at the end NEXT END STEP VARIAB FULLOUT PCRIT 2 END TEM eee eee s MANUAL 46 Juli Caujap Castells and Mario Baccarani Rosas STEP HDYWBG LEVENE EXACTP END STEP SIMDIS ALLCOEF SINGLE 2 END STEP COEFOUT BELOW 1 ABOVE 2 END STEP SINGLE COEF 1 END STEP DISTRIB COEF 8 END STEP FSTAT OUTPUT 1 END STEP WRIGHT78 END STEP WRIGHT78 NOHRCHY END STEP HETXSQ CONTAB SUBDIV 1 END STEP CLUSTER COEF 1 COPHEN COEF 9 END If you wish to remove commands or add new calculations just do it removing or typing lines in this ASCII file 3 2 THE BOTTLENECK FORMAT The program Bottleneck Piry Luikart and Cornuet 1998 applies a sign test for heterozygosity excess Cornuet and Luikart 1996 to detect whether the populations have experienced recent historical bottlenecks This test compares expected heterozygosity He under oe eee eee s Juli Caujap Castells and Mario Baccarani Rosas Hardy Weinberg expectatio
24. as before but with the number of columns equalling the number of groups you defined 3 Label the groups with proper names 3 Tick the cell s corresponding to the population s you want to include in each group In 7ransformer 2 a group can consist of any number of populations one population can be a group and one given population can appear in more than one group at the same time In the sections below we discuss several possible options to define population groups 2 2 1 Analysing population subsets Suppose you have a data set for a large number of populations but you are only interested in analysing only a certain sub group of populations within it This is how to do it a Write the number of groups you wish to establish in the corresponding cell and then press Return In the example below suppose we want to analyse only the five populations POP1 POP2 POP4 POP5 and POP6 Therefore we first have to write a 5 in the cell Populations After pressing TEM eee eee s MANUAL 39 Juli Caujap Castells and Mario Baccarani Rosas Return the original 7x7 matrix has changed into a new 7x5 matrix see Figure 32 Selecting Populations Ka tomm om om me mj m mi o POP POP2 POP3A POP3B POP4 HEHEHE amp HEHEHE M Figure 32 b Write the names of the populations you wish to include in this partial analysis in the column headers In the example Figure 33 we write these names in the
25. astells and Mario Baccarani Rosas WARNING FOR WINDOWS XP USERS If you are using Windows XP the macros will be probably disabled by default As Transformer 2 uses macros you will have to change your macros security option from high to medium in Tools Macro Security if you want to run the program TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas Section 1 Entering data Transformer 2 offers a versatile interactive data entry interface that makes corrections and manipulations easy to implement At present you can feed 7ransformer 2 with the drawings of the interpretations of your molecular patterns see section 1 1 or with a matrix of genotypes that you have to type see section 1 2 1 1 GENERALITIES ON THE DRAWING UTILITY Transformer 2 allows you to store your interpretations in an interactive drawing matrix This tool allows the user 1 To have a permanent record of the interpretations of molecular patterns that can be easily modified and corrected 2 To generate a genotype file for any combination of loci which is the basis for any subsequent data transformations and analyses Although we believe that drawing interpretations is advisable in most cases particularly if using allozymes it is especially so if you begin to interpret your molecular patterns right when you obtain the first consistent data Building your database little by little is practically effortless and allows you to
26. cells above the columns of the matrix Selecting Populations xu A E ed HA a POP2 POP4 POPS E E m Im El E E w Figure 33 de ebe MANUAL Juli Caujap Castells and Mario Baccarani Rosas b Tick on the cells of the new matrix corresponding to the populations you want to analyse under this data configuration In the example we have to tick only the cells that correspond to the populations we want to analyse Figure 34 Selecting Populations x A 5 Poplrene Genestat Biosys NTS s5 pe GenePop Bottleneck Prob Loss EXIT POP1 POP2 POP4 POPS POPE POP4 POP POP3A POP3B POP4 POPS POP6 BEEBE TLL BL HL Re L EL Ts HHH GR GR KN Figure 34 C Press the button corresponding to the transformation s you want to perform for that conformation of your data give adequate names to the files that will be generated and press save see section 3 After this 7ransformer 2 is ready to perform the calculations and obtain the formats for that set see section 3 2 2 2 Comparing independent groups of populations Most of the times understanding the genetic relationships among the organisms we are analysing entails the comparison of groups defined using different criteria of interest i e geographic distribution specific ascription habitat clade ascription etc Transformer 2 allows the user to establish groups within the data in the following way TEM eee eee s MANUAL 41
27. ct the option Current sheet in the button Format in the toolbar menu see Figure 7 Format Records Ea ll sheets Figure 7 Formatting a newly created sheet for drawing new enzyme primer interpretations Be careful not to select All sheets within Format unless you want to erase the whole contents of your interpretation file 1 1 5 Phantom bands and missing data Every locus colour in 7ransformer 2 has two associated degraded tones both of them fainter than the corresponding allele colour TEM eee ee ee s MANUAL 15 Juli Caujap Castells and Mario Baccarani Rosas that can be used to draw bands that you do not want to include in the final interpretations Figure 8 see section 1 2 8 for details EE GEEEEEEEEEEEEEEEEEEEEEEEE CR Figure 8 Example of phantom bands in a gel corresponding to the enzyme PGM There are phantom bands in the three loci defined for this enzyme see section 1 2 8 for details Also if you cannot interpret a given individual for a given locus you can leave it blank 7ransformer 2 will just add a 999 to the corresponding genotype when it interprets the pattern the file transf draw xls contains many individuals with empty loci 1 2 DRAWING YOUR INTERPRETATIONS 1 2 1 Getting started a Select the option Species population of the button Add in the toolbar menu Figure 9a 4 Move Left Insert P Delete Y Heterodimer Add Format Rec
28. d all the alleles in the desired positions Figure 14 illustrates the end of this process for the default pattern in Figure 13 di Move Left E Move Right A Heterodimer Add Format Records JO Pattern Interpretation a ET o ES oN Om CD ew Ne FE REl PER PE mi S wo 9 xo Figure 14 Modification of the pattern in Fig 13 obtained by moving the alleles to the right The allele movement began with the black allele labelled E followed with the one labelled D and so on until the red allele labelled A To insert or delete columns within the drawing matrix a place the pointer at the chosen place in the matrix and b press the button Insert column or delete columns as needed in the bar chart menu Figure 15 t Insert UW Delete Figure 15 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas Adding columns is adequate if you need a bigger matrix for drawing the interpretations of your molecular patterns new columns will be created at the right of the selected cell Deleting columns is an option you may want to take in order not to assign more space than strictly needed to represent your interpretations of a given Enzyme primer see Figure 16 However the minimum number of columns in an enzyme primer is 11 Once you reach this limit 7ransformer 2 will not allow you to delete more columns see section 1 1 2 3 You can add or delet
29. d from the file transf draw xls VERY IMPORTANT If you have drawn enzyme primer patterns that you did not interpret you must de select them from the corresponding loci found box associated to your project Transformer 2 does not distinguish if a locus has been interpreted so it would include the uninterpreted patterns by default in the genotype file and this would generate defective files 1 2 11 Tips on the drawing utility 1 Check carefully the position and colour of the alleles before generating the corresponding genotype file Remember that in order for an allele to be genotyped its position and colour must correspond to one of those defined at the enzyme primer header Otherwise 7ransformer 2 will not consider it 2 Take care not to draw more than two alleles per individual at a given locus However if you do so 7ransformer 2 will prompt an error message when you invoke the Pattern Interpretation command see section 1 1 8 You have to correct the mistakes that 7ransformer 2 will eventually pinpoint before moving on to the next interpretation TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas 3 he enzyme primer patterns you interpret must be in the active sheet If you introduce the code of an enzyme primer that appears in another sheet of your project 7ransformer 2 will tell you that it cannot find that item in the current sheet 4 If you want to change the position of one or several al
30. de Sample B L L6 1 2 oficinais POPI E L 2 oficinas POPI 2 E E L8 3 2 oficinais POPI 3 E E O AA BD 9 4 amp eem POPI 4 BN B EN 44 CE 110 5 amp oficinais POPI 5 E LP AA BD 11 86 Aficas POPI e E a N AA BD 1181 7 A oficinais POPI 7 E B B 4A AE 113 8 2 oficinais POPI 8 E E 44 CE 14 9 R oficinais POPI 9 E UN cc BO 115 10 2 oficinais POPI 10 E E N AA BD 16 11 oficinais POP2 11 B E 8 AA BD AR 12 2 oficinais POP2 12 E E E AA BD 18 13 oficinais POP2 13 B BN E WB c CE a9 14 ew POP2 14 E E AA EL 20 15 amp oficinais POP2 15 B B WT CE 121 16 amp eove POP 16 E E NM AA BD Paal 17 Rois POP2 17 B E WB 4A AE 23 18 2 oficinais POP2 18 HA H AB cc Figure 26 Genotypes appear after pressing OK in the enzyme primer interpretation box oe eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas If there are missing data for any of the individuals 7ransformer 2 will just assign a 999 to the corresponding genotype If you have drawn more than two alleles in an individual at the given locus 7ransformer 2 will warn you see the error message below so you can correct it before carrying on Enzyme Primer Interpretation Error Found on sheet 2 row 12 Press Ok to continue If you do not correct the mistake s the program will continue running but you will probably generate defective files or data see section 2 Therefore you are stron
31. e columns at any point of the interpretation process Be careful not to delete a column where you defined an allele Just in case 7ranformer 2 will always ask you to confirm the deletion before proceeding Figure 16 shows the effect of eliminating the spare columns at the left of the first allele of the red locus in Figure 15 4b Move Left x Mowe Right E bet P Delete Adda Formal Records Par Figure 16 Modification of the pattern in Fig 14 obtained by deleting columns at the left of the red A allele For a better visualisation of the patterns it is advisable to leave at least one blank column between consecutive alleles see Figure 10 However 7ransformer 2 does not have any problem with interpreting contiguous alleles not separated by a blank column see Figure 17 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas DE ABC DE AFBGCD Figure 17 Example of allozyme loci with several contiguous alleles for the enzyme PGM 1 2 5 Draw the alleles There is just one possibility to draw an allele for a given individual 1 select the cell where you want that allele and press Alt and X simultaneously DO NOT draw alleles by copying the coloured cell from the enzyme primer header and pasting it in the corresponding individual 7ransformer 2 will let you do it but this can give rise to errors in the subsequent interpretations Just use the described combination of keys If you use T
32. enePop see section 3 3 GeneStat PC 3 31 see section 3 4 NTSYS pc 2 02j see section 3 5 PopGene version 1 32 see section 3 6 Furthermore 7ransformer 2 calculates the probabilities of loss 4 sensu Bengtsson Weibull and Ghatnekar 1995 see section 3 7 and a table of allele frequencies associated with the configuration of populations that you have defined button Prob Loss 3 A square matrix where the rows and columns are the populations that are included in the genotype table 7x7 in the example Every cell in this matrix there are 49 cells in the example can be selected in order to define different configurations for analysis see section 2 2 The limit for the number of populations is 50 If you choose any calculation option for this default configuration the resulting analyses or data files that 7ransformer 2 will generate will correspond to considering all the populations individually EE MANUAL 38 Juli Caujap Castells and Mario Baccarani Rosas 2 2 DEALING WITH GROUPS OF POPULATIONS Many times the population geneticist is interested in obtaining the values of the genetic polymorphism parameters for different groups in which the data can be subdivided This utility of 7ransformer 2 consists of four basic steps 1 In the box of population selection choose the number of groups populations to be defined 2 Press Return Then the default matrix will reduce to a new one with the same number of rows
33. eo gen tico de poblaciones naturales de plantas vasculares Jard n Bot nico Canario Viera y Clavijo Cabildo de Gran Canaria Cornuet JM Luikart G 1996 Description and evaluation of two tests for detecting recent bottlenecks Genetics 144 2001 2014 Lewis PO 1993 GeneStat PC 3 31 North Carolina State University Raleigh North Carolina Luikart G Cornuet JM 1998 Empirical evaluation of a test for identifying recently bottlenecked populations from allele frequency data Conservation Biology 12 228 237 Oliva Tejera F Caujap Castells J Naranjo Suarez J Navarro D niz J Acebes Ginov s JR Bramwell D 2004 Variaci n gen tica de los Zotus L Fabaceae Loteae de pinar de Gran Canaria Bot nica Macaron sica 25 31 52 Piry S Luikart G Cornuet JM 1998 Bottleneck a program for detecting recent effective population size reductions from allele frequency data INRA URLB Laboratoire de Mod lisation et Biologie Evolutive Montpellier France Raymond M Rousset F 1995 GenePop version 1 2 population genetic software for exact tests and ecumenicism Journal of Heredity 86 248 249 website http www cefe cnrs mop fr TEM eee eee s MANUAL 26 Juli Caujap Castells and Mario Baccarani Rosas Richter TS Soltis PS and Soltis DE 1994 Genetic variation within and among populations of the narrow endemic Delphinium viridescens Ranunculaceae American Journal of Botany 81 1070 1076 Rohlf FJ 1998
34. esent in lt 50 of the populations considered b The other option that we will call Select enables the user to choose the alleles for these calculations by typing a 1 in the column labeled Select Figure 44 ALLELE freq Hobs Lo Lar Jeg Lo Joglo Select MDH1A 0 5252 D d 0 SPA EM UR NIDIH ID DIE d 0 Dam DUE 0335 1 ADH 0 188 0 0542 00842 1266 128 UR MIDH 2 amp 00536 5 0541 014231 0 666 03735 n MIR AR 1 1161 Pur Dir DIOU uu LN ADH de Z 00002 00002 2356 3 59 UR SIDH 2D 0125 7 04542 0 1542 0612 0812 n MDH 7E 1 453 00161 0061 1 75 175 LA DHIA nra 1 05452 O8F41 nno 01713 1 DHIE Danes 7 a 0 10 0632 10 0532 a DAL HES 2 042 0423 03 UE UR Drei 0 AO d Ur OFF DOS 26 LN DHIE 01633 1 016569 O088F 0 1758 1 UR TIA 05107 6 00002 3611 4445 n 1 18 0 Ane A 006 Dd 1571 7 l DR TiC 0341 Z DIR 00 8 15683 15233 D TAD 0 78 i 00625 0 12041 84288 DR BOILA Hi A Un UI USER 1 DR T 2H 03112 7 il O 147221 14 73 D Mi 0 1687 3 paa 00773 0 4781 1 1085 DR GHEE 0317 s UE 00012 6 22278 it Wi DA T OJ UI ee A LN Mi 03508 4 00015 00024 15015 UR bi O16 05658 0 4 00141 0 0833 1 D 2 07541 051 4 11 LES 1 Figure 44 After choosing the option Prob Loss a table like this one appears below the Table of allele frequencies By default the alleles selected are only the ones that fulfill the conditions described above under the name Viera y Clavijo However you can make your own selections by typing 1 on the column Select for a
35. esponding data file 4 Press OK 5 Your data are ready to run in GenePop 3 4 THE GENESTAT FORMAT GeneStat PC 3 31 Lewis 1993 calculates polymorphism indices gene diversities genetic distances and Nei s 1973 population structure statistics Hs Hz Js and Gei 3 4 1 Obtaining the GeneStat format 1 Press on the button GeneStat in the dialog box 2 Give a proper name to the corresponding data file 4 Press OK 5 Your data are ready to run in GeneStat 3 5 THE NTSYS FORMAT Ntsys pc 2 02j Rohlf 1998 is a multivariate statistical program that can be used for certain molecular population genetic data analyses It consists of several different modules and most procedures require the use of one or several of them The most frequently used options in Ntsys by the population geneticists are the genetic distance calculations clustering multivariate analyses and Mantel tests oe eee eee s MANUAL 49 Juli Caujap Castells and Mario Baccarani Rosas 3 5 1 Ntsys PC format requirements There are various entry formats in Ntsys For allele frequencies Transformer 2 generates three files that Ntsys requires for this kind of data 1 A data file with the allele frequencies 2 A sample size file 3 A locus size file 3 5 2 Obtaining the Ntsys format Transformer 2 gives you the formats for the three different files required to run allele frequency data in Ntsys To obtain these files for any of your populations
36. etics laboratories at the Jard n Bot nico Canario Viera y Clavijo since 1999 until present and a Ram n y Cajal researcher in this institution since 2001 until present Transformer 2 has been programmed by Mario Baccarani Rosas and is the result of a collaborative effort between the Jard n Bot nico Canario Viera y Clavijo and the Departamento de Ingenier a del Software of the Instituto Tecnol gico de Canarias ITC The Transformer project received support from the Cabildo Insular de Gran Canaria the Ministerio de Ciencia y Tecnolog a MCYT and the research projects REN2003 07592 GLO MCYT and Pi2003 032 Direcci n General de Universidades e Investigaci n del Gobierno de Canarias TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas GENERAL CHARACTERISTICS OF 7ZRANSFORMER 2 Transformer 2 is programmed in visual basic using a Microsoft Excel sheet so it will run in any computer that can contain the Microsoft Office package This program is suitable for codominant allozyme or microsatellite data for at least 60 enzyme primers with up to 10 loci per enzyme primer each locus containing a maximum of 10 alleles in 66 000 diploid individuals DISCLAIMER Transformer 2 can be downloaded from http www step es jardcan in the link Gen tica de la Conservaci n without charge and may be distributed freely if and when i it does not undergo any modification ii this manual and the two
37. f you want to insert a phantom band in a position which is defined as an allele in the enzyme primer header then a Select the position where you want to insert it and b Press ALT and C simultaneously oe eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas A degraded version of the corresponding locus colour will appear in the selected cell If you press ALT C again then an even fainter version of the locus colour will appear If you press ALT C a third time then the first degradation of the allele colour will appear and SO On 1 2 8 2 If the phantom band is not in a position defined as an allele in the enzyme primer header then see Figures 22a to g a Select the position where you want to place the phantom band in the enzyme primer header Fig 22a b Press Add a and then Allele in the toolbar menu Fig 22b C Press none in the dialog box that will appear Fig 22c d Select the corresponding degraded tone you want to assign to the phantom band Fig 22d and e e Draw the phantom bands Fig 22f and g a b C d kr d Hess er Ah e Fund ZA agin Hala de banal Baum dete 4 Hetarcciner Acd Forrat Pecords wet iss do ee oer Akn cm Zeck E A J D PEJ TEHERENEROHSERGTE 333332233323 3323 X DEP dele L oun TFT CESTO II TE RARA CARE ERRE A gn d rovwm rrmer SKD Gul eee e SKI w pa laziPo ladcn ANI in 1 A
38. gly adviced to correct any mistake before passing on to interpret the next pattern Once you have completed this process for all the enzymes primers included in your interpretation a Click on the button Genotype file in the toolbar and then select the option From the drawing Figure 27 Genotype File From the Drawing E E Input Data Figure 27 b Select the loci for which you want to generate the genotype file by ticking on the appropriate boxes default is all loci see Figure 28 and click OK Transformer 2 will then generate a genotype sheet that is the basis for the subsequent calculations and data transformations If you want to save this genotype workbook do it now The file transf gntp xls was obtained by invoking Genotype file for all loci in the file called transf draw xls TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas Creating Genotype File wl Loci Found Iv MDH 1 M MDH 2 M IDH 1 Iv GOT 1 Iv GOT 2 Iv PGhi 1 M PGM 2 DG hf EST 1 M EBT 2 M EST 3 Iv ME 1 Iv PG Iv PGI 2 M SKD 1 M SKD 2 Iv ADH 2 Iv FDH 1 Iv FDH 2 Iv FDH 3 Iv FDH 4 Iv TPI 1 Iv TPI 2 Iv TPI 3 Iv TPI B CANCEL I B PGD 1 M SKD 3 Iv TPI 4 M 6 PGD 2 Iv ADH 1 Iv TPI 5 Figure 28 Box to select the loci you want to include in the analyses By default 7ransformer 2 selects all loci in the project whose patterns have been interpreted obtaine
39. inalis a AC EE 38 me POP3B 38 B i AA CE 39 me POP3B 39 B E AC DD 40 Gef POP3B 40 B i AA CE 41 Gom POP3B 41 B BN lil AC Gt 42 mof POP3B 42 B BN E AC EE 43 Ao POP3B 43 B E AA cc 44 Go POP3B 44 B BN E AC cc 45 mo POP3B 45 B H AA EE 46 Go POP3B 46 A E CC EE AT amp officinalis E E 0E AA ED Figure 19a Select the position where you want to define a new locus b Choose Add and then Locus in the toolbar menu see Figures 19b and 19c d Move Left s Move Right 4 Insert H Delete Dimeric Add Format Reco ei A allele EF CHI JRERNCRGESTUNNDO LTTEEEEENM On Enzyme Primer M DH Se Specie y Sheet m ao d 37 amp officinalis 38 A officinalis POP3B 38 39 Aoc POP3B 39 40 Aoc POP3B 40 41 moves POP3IB 41 42 moe POPS3B 42 43 Gom POPS3B 43 44 move POPS3B 44 45 Gom POPS3B 45 46 move S POPS3B 46 4 A officinatis E EB NN NND NND eee H EB NN NND UNS UN m mm Figure 19 b Select Add and then locus from the toolbar menu TEM eee ee ee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas Move Left Move Right 6 Insert HF Delete Dimeric Add A E E A A A FF Eo E MM 38 ovas POP3B 38 B E E 39 mew POP3B 33 B E 40 aos POP3B 40 E H BH 41 move POPE 41 B B A 42 orina POPIE 42 E B 43 mew POPE 43 B E Hora POP3B 44 B B 45 Goose POPE 45 E A 46 oca POPE 46 H
40. is an allozyme with uncertain quaternary structure it is represented as a dimer TEM eee ee ee s MANUAL 23 Juli Caujap Castells and Mario Baccarani Rosas You can draw any number of these phantom bands as Transformer 2 will not interpret them remember it only interprets the alleles whose colours and positions coincide exactly with those defined as alleles at the enzyme primer header 1 2 9 Inserting new individuals You can add new individuals to the drawing matrix at any point of the interpretation process The only small restriction is that if the newcomers belong to a population that already has representatives in the drawing they must be added after the last individual for that population If a new population is to be added in your project then Transformer 2 will do it at the end of the existing file select Species population in the button Add on the toolbar menu and carry on as described in section 1 2 1 To insert individuals in an already existing population a Select any individual in the population where you want to add the new samples see Figure 23a b Click on the option Add in the button Records on the toolbar menu see Figure 23b C Just in case 7ransformer 2 will remind you you re just about to add new individuals in that population see Figure 23c d Click OK and the pointer will move to the position where it will insert the first of the new individuals i e right after the
41. l gel shuffled back to front and then turned 909 counter clockwise see Figure 1 for an illustration 1 E NE EHE ont y nun E I H 1234556 738mm 6 P E E BN BN el TT c E 9 EHE E EHE E Figure 1 Original picture of an allozyme gel for a monomeric enzyme left and how should it appear in the drawing matrix of 7ransformer 2 right Different colours stand for different loci red is locus 1 and black is locus 2 Although this way of drawing may appear counter intuitive at first it does not take long to become familiar with it Its advantages are that it allows the program to have faster analytical algorithms while TEM eee ee ee s MANUAL 11 Juli Caujap Castells and Mario Baccarani Rosas the user can read the alleles from left to right in several loci for many individuals 2 For each enzyme primer the drawing utility of 7ransformer 2 consists of see Figure 2 a an enzyme primer header that contains i the name of the enzyme primer framed and ii the positions of the alleles detected b a drawing matrix where you can insert and delete columns to make it fit your molecular patterns C a genotype area with the label Gntp containing as many columns as loci you have defined for that enzyme primer the limit is 10 loci per enzyme primer These columns will be coded according to the enzyme primer name and will remain empty until you decide to genotype that locus see section 1 2 10
42. leles after completing the process of pattern interpretation you can do it but you will have to press pattern interpretation for the affected loci and then genotype file again Otherwise the genotype file will be the same as the one without the change s 1 3 ENTERING A TABLE OF GENOTYPES Users of 7ransformer 2 that already have genotype matrices for their data may rightly consider that drawing their interpretations would be burdensome and time consuming For such cases Transformer 2 offers the option of entering a table of genotypes 1 3 1 How to input your genotypes for analysis In the 7ransformer 2 toolbar menu Select Input data on the option Genotype file Figure 29 Genotype File 7 j From the Drawing OE Figure 29 Selecting the option to enter a genotype Table in 7ransformer 2 Then you just need to have a Microsoft Excel genotype table like the one in the attached file transf gntp xls see Figure 30 for an example In this file a The first line should contain the headers for the species populations and loci In this line the first column is the sample number the second is the species name and the third is the population code Only make sure that the string of letters in the population code TEM eee eee s MANUAL 35 Juli Caujap Castells and Mario Baccarani Rosas column is exactly the same for all the individuals that you want to include in a given population and follows the
43. li in contact with the researchers at the Divisi n de Software of the Instituto Tecnol gico de Canarias ITC who were very receptive at the idea of helping develop a better Transformer After several meetings the project was undertaken by Mario Baccarani Rosas who is the programmer of 7ransformer 2 and has made possible many ideas that were just starving for opportunity In 7ransformer 2 a lot of effort has been devoted to the entry formats especially in the drawing matrix and to the configuration protocols with the purpose of making the most of the data in the shortest possible time We believe that the use of the program is quite intuitive and user friendly Probably 7ransformer 2 will be especially welcome by those working with allozymes though it can also be used with microsatellite data Its versatility that we hope to enhance much further very soon can save a lot of research time and avoid most errors associated with genotyping formatting and data analysis of molecular population genetic information Juli Caujap Castells and Mario Baccarani Rosas Las Palmas de Gran Canaria August 2004 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas INDEX WARNING FOR WINDOWS XP USERS Section 1 Entering Data 1 1 Generalities on the drawing utility Ill Advantages of drawing the interpretations General features of the drawing matrix Drawing heterozygous individuals Adding sheets to your drawing project Pha
44. n D E na Mis T ON t E A Chest ampie NM BE E 21 IT i be coon lum nr i r H r E E D E a B OC A E D E n E E 8 a 3 H H u EE Hsi M Cula vc camp n 1 E 8 A i i i D E D Bases At TI i T 1 Inscr Allcle x EBEN m m an A 8 8 an u a u WISBUPBENTN SD TE Eug P deaur 1 mt Wm Cum sma Wu Pre nu a 2 nu a E This new allele will be assigned to locus Sie carers quer terra Wt can C au A A at H H LE I B a E at H H H L H H E H E E a HONZ mmi A a a H H H L E a a i a H D E E e e i CANCEL E E A E E E Mou 5 lc maka mak ane at contes t f 2 mar lo be MATOS re is Magis ns nre af cis cc enm vb ee ost lg ele A liste oder eka Lea kee QU ete 4 licterccie Add cima Jee lleterodmer Add loin ACCA D cp c AD c B c2 E E c BIHE H om E EN 4 2 EIE BN EE UN E E E L A A D 2 E E A Casi ale e Colcrs E E A d il u DH B E DH E Ne EG P722 scele TT e ze coe sume Fer Fame L H a a A E Mor nw tant qur orit bons rne dnas a ia H a an H H RI D E a A A H nn E A EE A A nt n va rt A A H A E B H B Em in E E nu E 1 nn u il la LE A A B i H a E H L E ur LIU riske gair AE Jn us der eu Ls mt do be E E B A E E Y Y elec Fatz amr A be E D E E E a a E E E E B B pl A Figure 22 Drawing phantom bands in a position not defined at the enzyme primer header In the example although SKD
45. n the dialog box with the default name ntsys samplesize Figure 43 just re name it as you wish Creating NTSYS Sample Size File 2 x Guardar en 3 Mis documentos D t a Ke Ci v Herramientas Ly Corel User Files SA Mis im genes Historial 9 My Archives My eBooks a all nts a freq nts TERRE 12 ntsys_alleles nts a ntsys frequencies nts EI sample nts Favoritos Nombre de archivo TEENS Eder Guardar como tipo ursvs Fies Datei E Cancelar Figure 43 4 Press OK 5 Your data are ready to run in Ntsys TEM eee eee s MANUAL 1 Juli Caujap Castells and Mario Baccarani Rosas 3 6 THE POPGENE FORMAT PopGene version 1 32 Yeh et al 1997 is a program for the analysis of co dominant and dominant diploid and haploid molecular markers It calculates most basic parameters of population genetic variation for different types of population structure and allows the user to perform many tests bearing on the structure of data i e the homogeneity test Ewens Wattersson neutrality tests and the two locus linkage disequilibrium test 3 6 1 Obtaining the PopGene format To obtain the PopGene file for any configuration of your populations do this 1 Press on the button PopGene in the dialog box 3 Give a proper name to the file 4 Press OK and your data are ready to run in PopGene 3 7 THE PROBABILITIES OF ALLELIC LOSS Rare alleles are important in Conservation Biol
46. nds Spain JARDIN Jardin Bot nico Canario Viera y Clavijo BOTANICO Laboratorio de Biodiversidad Molecular CANARIO Ap de correos 14 de Tafira Alta VIERA Y 35017 Las Palmas de Gran Canaria CLAVIJO Canary Islands Spain Cana Sg Si a Instituto Tecnol gico de Canarias D Departamento de Software En ae o BIT C Playa de Pozo Izquierdo s n e a 35119 Santa Luc a Las Palmas de Gran Canaria o aa Canary Islands Spain t 03n ve a a E MANUAL Juli Caujap Castells and Mario Baccarani Rosas ON THE Transformer PROJECT AND 7ransformer 2 The Transformer project aims at streamlining the generation storage interpretation processing and application of molecular population genetic data especially as related to Biological Conservation Transformer 2 is one computer program within the Transformer project It allows the user to concentrate in the accurate interpretation of molecular patterns and in the discussion of quantitative results through automating data transformations and analyses that are otherwise burdensome complex and prone to error Through saving research time while increasing accuracy Transformer 2 permits the effective implementation of urgency in the growing number of practical applications of molecular population genetic information CREDITS The Transformer project was conceived and developed by Juli Caujap Castells while he was responsible for the molecular population genetics and phylogen
47. ns to the heterozygosity expected at mutation drift equilibrium Heg in a sample that has the same size and the same number of alleles as the sample used to measure He Luikart and Cornuet 1998 The rationale of the test is that since low frequency alleles are lost at a much faster rate than heterozygosity in a bottleneck situation bottlenecked populations are expected to have a heterozygote excess 3 2 1 Obtaining the BOTTLENECK format The Bottleneck option in 7ransformer 2 gives you a single file that contains the format for all the populations or population groups in the configuration that you defined To obtain it 1 Press on the button Bottleneck in the dialog box 2 Give a proper name to the corresponding data file 4 Press OK 5 Your data are ready to run in Bottleneck 3 3 THE GENEPOP FORMAT GenePop Raymond and Rousset 1995 is a software package that runs under the DOS operating system The DOS version is updated periodically and contains a few options not available on the web site of the program website http www cefe cnrs mop fr GenePop allows the user to perform most calculations and tests related to the estimation of population genetic variation from the information contained in molecular markers 3 3 1 Obtaining the GenePop format 1 Press on the button GenePop in the dialog box TEM eee eee s MANUAL 48 Juli Caujap Castells and Mario Baccarani Rosas 2 Give a proper name to the corr
48. ntom bands and missing data ing your interpretations Getting started Choosing population codes Define the enzyme primer Place the alleles in their correct positions Draw the alleles Inserting new alleles in the drawing matrix Inserting new loci Drawing phantom bands Inserting new individuals 10 Get the genotypes from the drawing 2 11 Tips on drawing the data 1 3 Entering a table of genotypes 1 3 1 How to input your genotypes for analysis Section 2 Configuring the data 1 The default configuration 2 Dealing with groups of populations 2 2 1 Analysing population subsets 2 252 Comparing independent groups of populations 2125435 Including populations in more than one group Section 3 Processing the data 3 1 The Biosys format 3 1 1 Obtaining the Biosys format 3 2 The Bottleneck format 3 2 1 Obtaining the Bottleneck format 3 3 The GenePop format 3 3 1 Obtaining the Genepop format 3 4 The GeneStat format 3 4 1 Obtaining the GeneStat format 3 5 The Ntsys format 3 5 1 Ntsys format requirements 3 5 2 Obtaining the Ntsys format 3 6 The PopGene format 3 6 1 Obtaining the PopGene format 3 7 The probabilities of allelic loss 3 7 1 On the probability of loss 3 7 2 Obtaining the probabilities of allelic loss 3 8 Tips on processing the data LITERATURE CITED APPENDIX The two example files 1 2 C dO UU bt S Ui NU NO NJ NJ NJ NJ NJ NJ NJ NJ NJ NJ E O Ze 2 TEM eee eee s MANUAL Juli Caujap C
49. ny allele In this Table freq is the average allele frequency in the chosen group Nobs is the number of populations where the allele was detected and Lo and Le are the observed and expected probabilities of loss ve ee eee ees MANUAL Juli Caujap Castells and Mario Baccarani Rosas By default 7ransformer 2 obtains the value of both the expected probabilities of loss Ze i e assuming that the allele had its overall average frequency at each of the populations considered and the observed probabilities of loss Lo for all the alleles that fulfill the Viera y Clavijo conditions If you press the button Create chart that appears below the Table of the probabilities of loss Figure 45 the values of L and Ze are used for two linear regression analyses Bengtsson et al 1995 where the average frequency of each allele is the x axis and log Lo and log Ze are the respective y axes Figure 46 The chart is created only for the alleles that are selected in the column labeled Selection TPEFSB 0 361 Fl 0 QO 19 229 19 7229 A TPLSC 0 04b 2 055 051 00017 0 206 1 TPI BA 0 3261 5 00193 0004 1 713 2 3992 0 TPFBB 0 7671 El 0 605 005 A Figure 45 The button Create chart appears at the end of the table that contains the probabilities of loss 19 Alleles R 0 298 c Cl 5 co Oo HE On log Lo O log Lel co Oo R3 tu log Probabilities of loss a na 0 D 02 O04 0 06 0 08 AN
50. ogy because they represent unique evolutionary byproducts that may endow a species with advantageous properties to cope with eventual environmental shifts Schonewald Cox et al 1983 Richter et al 1994 Bengtsson et al 1995 Thus collection designs oriented to sampling rare alleles provide the manager of genetic diversity with adequate tools with which to reinforce declining populations or aid the survival of reintroduced plants The probability of allelic loss facilitates a straightforward way to analyse rare alleles and to incorporate them into conservation practice Caujap Castells and Pedrola Monfort 2004 TEM eee eee s MANUAL 92 Juli Caujap Castells and Mario Baccarani Rosas 3 7 1 On the probability of loss Transformer 2 calculates the probability of loss Z i e the probability that a sample of size W fails to include an allele with population frequency p using the expression Bengtsson et al 1995 L 1 py Because these calculations are only suitable for alleles that are rare in some way Bengtsson et al 1995 and there is no universally accepted definition of rarity 7ransformer 2 offers two options to select the alleles for the calculation of a The default option that we will call Viera y Clavijo follows Caujap Castells 2004 Caujap Castells and Pedrola Monfort 2004 or Oliva et al 2004 and calculates Z only for the alleles that 1 have an overall frequency lt 0 5 and 2 are pr
51. orc BXB M 999 A Allele mel L L Locus On Enzyme Primer j Sheet Figure 9a oe eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas b Introduce the name of the first species you want to include in the drawing file you are about to create the population code and the number of individuals in that first population in the dialog that will appear Figure 9b Name D officinalis Code POPI Quantity 10l OK CANCEL Figure 9b Introducing a population in the 7ransformer 2 drawing sheet Just feed in this box what you have at present and do not worry if you plan to include more populations in your project or sample more individuals for a given population you will be able to add these at any moment of the interpretation process see section 1 2 6 1 2 7 and 1 2 9 After filling in this box 7ransformer 2 will write automatically in the drawing sheet the number of individuals that you have assigned to each population using four columns see Figure 10 1 The first column is the total number of samples 2 The second column is the name of the species you have input 3 The third column is the population code of your choice 4 The fourth column is a numerical free code that you may want to assign in order to identify each individual Write only in the fourth column to introduce the individual codes It is better not to write anything in the other columns e Move Left A Insert YP
52. r 2 can deal with are HILL LAKE HILL1 HILL2 HILLA HILLB LAKE SP1 LAKE SP2 101 A 101 B Have a look at the attached file transf example xls for other examples 1 2 3 Define the enzyme primer After inserting the samples of your project you have to define the basic traits of your enzymes primers 1 Select enzyme primer in the button Add from the toolbar Figure 12a Then you will be presented with a menu that asks you to input the basic characteristics of the molecular patterns you re ve ee eee ees MANUAL 18 Juli Caujap Castells and Mario Baccarani Rosas about to introduce Figure 12b in order to configure the loci in the 7ransformer 2 drawing sheet Add Format Record A Alle L Locus on EnzvmeJPrimer Ze Species Population Sheet Figure 12a Adding an Enzyme Primer to the drawing sheet 2 Fill in the dialog If you are starting the molecular interpretations from scratch just feed the number of alleles you detected in your first gel Again do not worry about new alleles individuals or loci that you may have to add in the future you will be able to do it easily at any point of the interpretation process see sections 1 2 6 1 2 7 and 1 2 9 In the example in Figure 12b the enzyme MDH has two loci with 3 and 5 alleles respectively d Move lat M bet Gest db Wieder Ale Format Records 0 Pattern interprets ib Luci Characteristics cunt
53. ransformer 2 for drawing microsatellite profiles take into account that at present the program does not take size of the allele or number of motive repetitions as a variable so it will just assign an A to the smaller allele a B to the second smaller and SO On 1 2 6 Inserting new alleles in the drawing matrix Whenever you detect a new allele in a locus you have to define it first in the enzyme primer header or 7ransformer 2 will not recognise it as an allele see the sections 1 2 3 and 1 2 10 To define the position of a new allele in one of the already existing loci follow these steps a Select the cell where you want to place the new allele TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas You can choose any position in the space assigned to alleles in the enzyme primer it belongs see Fig 18a b In the box that will appear select the locus colour where that allele should be assigned Transformer 2 asks you this because there are no restrictions on the relative position of any allele within the drawing matrix for a given locus This means that for instance an allele of the first locus red can be placed in the middle of two alleles from the third locus blue as well as in the middle of two pre existing alleles for the first locus Whatever the case 7ransformer 2 will automatically recode the old alleles according to their new relative positions see Figures 18a to f for examples
54. rt the heterodimer its symbol can be drawn in one of two ways a pressing simultaneously Alt and Z or b pressing the button heterodimer in the bar chart menu a LEN B a RR Lu HE LEN H HE EE H HE EHE HE HE LEN B H LEN B HE EE E A Lu He HE EE HE HE EE HE HE EE HE L LE Lu a LE a a i B LE E HE LE E Figure 5 Example of heterozygous individuals in three of the four loci defined for the dimeric enzyme FDH in the transf draw xls example file the black locus FDH 2 is monomorphic in this section of the file ve a a MANUAL Juli Caujap Castells and Mario Baccarani Rosas 1 1 4 Adding sheets to your drawing If you have many polymorphic enzyme primers in your project it is probable that their interpretations do not fit in a single Excel sheet Excel has a very short column number limit In this case you can add new sheets selecting the option sheet in the button Add in the toolbar menu see Figure 6 The first sheet will be named sheet 1 the next one sheet 2 and so on up to eventually sheet 10 Add Format Record A Allele 7 L Locus o Enzyme Primer CC Species Population Figure 6 Selecting Add sheet from the toolbar menu The contents of the newly added sheet will be exactly the same as that of the first one including the drawings You have to format the new sheet so that it only keeps the species and population codes for your samples To do this sele
55. sed NONE in Figure 18c a dialog like this would appear Although all possible allele colours are shown only selecting the black cell under Basic allele colours would insert an allele because the chosen enzyme ADH only has two loci defined If we choose a colour other than black an error message would appear lete Heterodimer Add Format Records ADH A ECA DE BC ns eff os NN 41 ADH Figure 18 f After pressing OK in 18 e a new allele appears at the black locus and the pre existing alleles at that locus change their codes according to their new position Remember that 7ransformer 2 only understands diploid data so that a maximum of two different bands with the locus colour can be used for genotyping an individual at that locus Just in case if you draw more than two alleles per locus in a given individual 7ransformer 2 will pop out an error message when it interprets the patterns see section 1 2 10 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas 1 2 7 Inserting new loci If you want to assign a new allele to a new locus that you had not detected in the previous analyses then just a Select the position where you want to place the new locus in the enzyme primer header like in Fig 19a Move Left s Move Right Insert UI Delete Dimeric Add Format Recc Genotypes A C E E a B MIDH 1 MDH 2 37 A offic
56. tenth whose alleles are violet see Figure 3 for the colour codes associated with each locus 5 Transformer 2 will only interpret the alleles in the drawing matrix whose colour and position are defined at the enzyme locus header see section 1 2 10 The palette of pre assigned allele colours for the maximum of ten loci is illustrated in Figure 3b Enzyme Primer OK Humber of Loci U CANCEL Loci Characteristics CODE COLOR H ALLELES Add Format Recorc A allele L Locus om EnzvmeJPrimer qi Species Population 1 Sheet ve ebe MANUAL Juli Caujap Castells and Mario Baccarani Rosas 1 1 3 Drawing heterozygous individuals Heterozygous individuals in monomeric allozyme loci and in microsatellite loci should be represented by two bands of the same colour see Figure 4 E NH NN NND NND NN ND ND ND ND UND ND UN E NN NN NND NND NND NON L o Figure 4 Example showing several heterozygous individuals in the monomeric enzyme SKD from the transf example xls file Note that only the red locus SKD 1 and the black locus SKD 2 have heterozygous individuals for this section of the data Heterozygous individuals in dimeric and multimeric allozyme loci should be represented by three symbols the two bands at the extremes should be assigned the corresponding locus colour and the heterodimer s should be a pre defined symbol see Figure 5 After selecting the cell where you want to inse
57. terns for SKD ADH FDH and TPI Rather than to provide the user with a real case these examples try to account for a panoply of possible situations that the population geneticist might be confronted with when analysing molecular patterns for diploid codominant markers Although most of the drawings are based on real patterns obtained for different Canarian endemics at the Laboratorio de Biodiversidad Molecular of the Jard n Bot nico Canario Viera y Clavijo these examples are a mixture that does not correspond to any real organism They also incorporate several locus configurations that were drawn on purpose to illustrate how 7ransformer 2 deals with particularly complex situations There are enzymes with just one associated locus and enzymes with many associated loci monomorphic loci ME 1 and PGI 1 moderately polymorphic loci and extremely polymorphic loci The patterns for the enzymes also contain missing data and phantom bands for different individuals at different loci and consider allele positions that would be particularly error inducing if the molecular patterns were interpreted or corrected by hand Use these files to make your first trials with 7ransformer 2 and to check the versatility of the program You may want to add new enzymes loci alleles or individuals and then try to perform calculations and generate data files for all the configurations of loci and populations you may think of This way you will get acquainted
58. track eventual changes and check previous interpretations easily while saving a lot of time and errors 1 1 1 Advantages of drawing the interpretations The major advantage of drawing the interpretations is that once you are done quantitative data for any possible configuration of populations and loci will be a few easy clicks away see sections 2 and 3 TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas However there are at least three more powerful reasons to use this tool of Transformer 2 1 You can forget about genotyping individuals as 7ransformer 2 will do it for you see section 1 2 10 Therefore you are less prone to make the mistakes that are so frequent when you interpret by hand 2 You may correct or modify your interpretations see sections 1 2 6 to 1 2 9 by moving inserting or deleting any number of individuals alleles loci or spaces easily at any point of the interpretation process 3 You will have a visual record of the interpretations which is much easier to scan than a whole table of genotypes and which can be used nicely in presentations see the attached file transf draw xls 1 1 2 General features of the drawing matrix 1 The drawing matrix of 7ransformer 2 is conceived to draw the interpretations so that the fastest alleles appear at the left hand side of the drawing and the slower ones at the right hand side To put it formally the drawing corresponds to the origina
59. ulations involved in the analysis of molecular population genetic data while keeping the versatility of the present version TEM eee eee s MANUAL Juli Caujap Castells and Mario Baccarani Rosas ACKNOWLEDGEMENTS We thank all the biologists and students at the Jard n Bot nico Canario Viera y Clavijo who were the first to provide data and feedback to improve the program especially Carolina Su rez Garc a Olga Fernandez Palacios Sara Mora Blas Vilches Felicia Oliva Tejera Magui Olangua and Juan Luis S nchez We also thank the people that have helped us through their continued friendship support and scientific stimulation especially Julia P rez de Paz Rosa Febles Alicia Roca Bernardo Navarro Pepe Naranjo Aguedo Marrero and Pepa Navarro at the Jard n Bot nico Canario Viera y Clavijo Miguel Gonz lez P rez and Pedro Sosa at the Universidad de Las Palmas de Gran Canaria Pilar Catal n at the Universidad de Zaragoza Jerzy T Puchalski at the Polish Academy of Sciences or Juan Mota at the Universidad de Almer a We are much indebted to Eugenio Reyes for his encouragement and for provoking the first contact between the Transformer project and the ITC Joaquin Oc n director of the Departamento de Ingenier a del Software at the ITC and David Bramwell director of the Jard n Bot nico Canario Viera y Clavijo are acknowledged for their willingness to allow the collaboration between these institutions
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