Tom H. Lindner & Katrin Hoffmann Manual easylinkage Plus v5.05

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2 The following programs were implemented under a graphical user interface: 1. FastLink v4.1 (parametric linkage analysis; 2. SuperLink v1.6 (parametric linkage analysis; 3. SPLink v1.09 (nonparametric single-point linkage analysis, particularly for affected sib pair designs (ASP); 4. GeneHunter v2.1r5 (single-/multipoint linkage analysis; 5. GeneHunter Imprinting v2.1r3/v1.3 (single-/multipoint linkage analysis; 6. GeneHunter TwoLocus v1.3 (single-/multipoint linkage analysis; 7. GeneHunter Plus v1.2 including ASM lin and ASM exp ( 8. Allegro v1.2c including simulation, and conditional analyses (Kong & Cox, Nat Genetics 1999; allegro@decode.is) 9. Merlin v1.0.1 (multipoint nonparametric linkage analysis; SimWalk v2.91 (multipoint linkage analysis; PedCheck v1.0 (test for Mendelian errors prior to linkage analyses; FastSLink v2.51 (single-point simulation program; ftp://linkage.rockefeller.edu/software/slink) GeneHunter, GeneHunter Plus, GeneHunter Imprinting, SPLink were recompiled (MingW) for the use in Microsoft Windows. FastLink, SuperLink, Slink, Merlin are available as DOS runtimes over the internet but will also be provided in our software package. PedCheck is available after free registration. Allegro can be obtained free of charge upon request from allegro@decode.is for academic users. Note that Allegro 2.0 is not supported! The same procedure applies for GeneHunter Imprinting/TwoLocus. However, the author of the Gene- Hunter Imprinting/TwoLocus extensions did not agree with providing windows runtimes for those programs in our setup package. The source code/executables) can be obtained from and must be manually implemented. If easylinkage cannot find Allegro, PedCheck, GeneHunter Imprinting/TwoLocus as stated in the INI file all options regarding those programs will be deactivated. This start-up check will be performed each time you call easylinkage so that the later addition of those programs will be noticed right away by easylinkage. No further user interaction will be required in this regard. easylinkage was tested under Microsoft Windows 2000/XP. It might run on older NT versions or Windows 95 but has not been tested. It could be that some source code changes have to be made, in particular to the routines with DOS commands. We thank Drs. Alejandro Schaffer (FastLink), Jurg Ott (SLink), Michael L. Frigge (GeneHunter Plus), Leonid Kruglyak (GeneHunter), and David Clayton (SPLink) for the permission to recompile the source code of their program for the use in Microsoft Windows and for publishing the binaries on our website. 2

3 License agreement IMPORTANT!!! There is a risk in running third-party binaries. Users are advised to compile their own binaries! LICENSING of Tom Lindner s/katrin Hoffmann s program for genetic analysis THIS SOFTWARE IS PROVIDED AS IS AND ANY EXPRESS OR IMPLIED WAR- RANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DIS- CLAIMED. IN NO EVENT SHALL THE AUTHORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAM- AGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTER- RUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE. Tom Lindner ( t-lindner@gmx.de) Katrin Hoffmann (katrin.hoffmann.genetik@charite.de) August 16 th, 2004 Friedrich Alexander University Erlangen Nuremberg Medical Clinic IV/4 Division of Nephrology, and Hypertension Community Hospital Nuremberg South Breslauer Str Nuremberg Germany And Institute of Medical Genetics Charité Berlin, Humboldt University Augustenburger Platz Berlin Germany 3

4 General notes easylinkage was designed to make the use of linkage programs user-friendly and to enable those analyses on Microsoft Windows based operating systems. The idea came up when the programmers had to run several linkage programs such as those that were implemented in easylinkage and tried to get their own linkage projects done. Very quickly it became obvious that all programs were not easy to handle at all. Most programs used different input formats. Appropriate files had to be generated in a time-consuming process in addition. No program provided a marker database from which the genetic positions could be drawn and used for automatic linkage analyses. Only a few programs were recompiled for running on Windows systems which is not really understandable since most marker genotypes were generated on such systems. Further, graphical outputs were almost completely missing. easylinkage overcomes all those pitfalls. Since the introduction of the program as version 2.01 major changes have been applied. In general, the user can analyze individual chromosomes or even entire genomes. The user can furthermore select between at least 5 different allele frequency algorithms that have to be used with care of course. For SNP projects reference allele frequencies are provided for Asian, African American and Caucasian populations (kindly provided by Affymetrix Inc.). Besides SNP projects, the user can run his analyses under the use of sexaveraged, female, or male marker map positions. Allele frequency algorithms were programmed universally for all subsequent applications. However, every algorithm has its limitations. The user should have an idea of which algorithm will deliver the best results. Here are the features of the possible algorithms: 1. Option Codominant : This option is the most commonly used option. Only individuals who are encoded by the pedigree information file will be used for allele frequencies computations. Only alleles occurring in genotyped individuals of the pedigree will be considered. After that frequencies will be set equally in order to sum up to 1. In the case the sum differs slightly from 1 the allele frequency of the highest number allele will be adapted. Otherwise some linkage programs will not run correctly. The routine is only used if the marker presents with more than 5 alleles. If lesser alleles occur 5 alleles are set with an equal frequency of In our experience this option is suited in particular for extended pedigrees. 2. Option All individuals from ABI file (example of a marker file at the end of the manual): This option considers all subjects within a genotyping file independent of their relationship to each other within a pedigree. If the pedigree information file contains many small nuclear families (like in affected sib pair designs) this option is reliable. The option is suited for ASP designs and delivers almost identical results when compared with the First person of an ASP option. 3. Option All individuals from pedigree file : This option considers only those genotyped subjects who are listed in the pedigree information file. This option reflects a slight deviation from the All individuals from marker file option. This option is suited for ASP designs. 4. Option Founders only : This option has to used very carefully. Only if the pedigree information file contains at least 10 founders this option can be used. Non-founder allele fre- 4

5 quencies will be set to If allele frequencies do not sum up to exactly 1, the frequency of the most common allele will be adapted. Dependent from the pedigree structure the number of non-founder alleles is more or less significant. The option can lead to a substantial deviation from true (population-based) allele frequencies. Situations can occur where many alleles are just missed. This option might be useful for some analyses. 5. Option First person of an ASP : This option is thought for ASP designs in particular. In this case only one genotyped subject per sibship is considered for calculating allele frequencies. However, this algorithm can miss alleles given by non-considered but genotyped subjects. In this case, the frequency of those alleles will be automatically set to In our experience results do not differ too much from the option All individuals from pedigree file. 6. Options Affymetrix Ref Asian / African American / Caucasian : Those options can only be used for SNP projects. Those projects refer preferentially to Affymetrix DNA SNP chips. Here, no specific allele frequency algorithm is used. Instead the user can choose between the three reference populations where the frequency for allele A will be provided. The database was kindly provided by Affymetrix Inc. In a case, where the frequency of allele A is this frequency will be set to depicting allele B still as being possible but at a very low frequency. In a case, where a reference frequency is not available easy- LINKAGE assumes an equal allele distribution. Some programs have trouble with certain family or subject identifiers. Therefore, easy- LINKAGE offers the opportunity to recode all identifiers to integers. Of course, after running the program the recoded identifiers will be decoded back for all generated outputs. Some programs do also have problems with non-continuous alleles (2, 10, 11 etc.). Recoding in continuous alleles can also be activated (1, 2, 3 etc.) and is strongly recommended. Programs tended to behave strangely if this option was not activated. In extension of the original version the program runs PedCheck for the identification of Mendelian errors prior running subsequent linkage programs. This option can be deactivated. However, the process is usually not very time-consuming. For microsatellite projects the user has to provide 2 types of input files: A pedigree information file containing the structure of one or more pedigrees in general linkage format (column 1: Family ID, 2: Subject ID, 3: Father ID (0 denotes a founder), 4: Mother ID (0 denotes a founder), 5: Sex (1 = male, 2 = female), 6: Affection status (= unknown, 1 = unaffected, 2 = affected)), and marker files containing the genotypes of the individual. Subject IDs have to be unique throughout the pedigree information file in order to match them correctly to the IDs in the marker files. The pedigree information file must contain an additional column 7 for DNA availability if the user wants to run single-point simulation studies (SLink). Marker genotypes have to be provided in individual marker files containing the marker name, the subject ID, and the genotypes in integer format. For SNP projects only two files are necessary: one file that contains all genotypes, one file with the pedigree information in linkage format. A datafile that contains the inheritance model is not needed anymore. easylinkage solves this issue internally in interaction with the user. All options can be clicked, edited, and set by using the mouse in specific option menus. 5

6 After starting the program all set options from the most recent analysis are reactivated automatically. The program remembers up to 20 directories in which analyses were performed earlier. Entry number 21 deletes the oldest entry. Each directory that is selected within the MAINSCREEN will be screened for pedigree information files immediately. Only files that start with p and end with pro will be recognized as pedigree information files. Whitespaces (spaces, commas, dots, tabs) are not allowed within the pedigree information file name. _ can be used. A major benefit of easylinkage is the generation of structured text outputs and graphical plots of LOD scores, P values, and many other parameters. Plots will be provided as chromosomal or genome-wide plots. All plots display details of the used inheritance model, marker map, sexspecific or sex-averaged marker positions, the number of known and unknown markers, in SNP projects even the number of uninformative SNPs, a table with the top five markers, the used pedigree file, date and time and elapsed time, directory, allele frequency algorithm, and other parameters. Plots can be generated as TOTAL plots averaging all families or plus individual family plots. The implementation of SNP projects derived from the Affymetrix 10k/50k/100k/500k and Illumina 5k chips is another major step ahead. Those projects can be analyzed with Allegro (single-/multipoint analyses), GeneHunter/-Plus, Merlin, FastLink, SimWalk and SuperLink. Some tricks have to be applied to enable the correct run of Allegro. Since all multipoint linkage programs assume linkage equilibrium between neighboring markers this preference can be easily broken by SNP data. Many SNPs are very closed to each other in terms of their genetic positions. If such a case occurs the program does automatically set the distance to a recombination fraction of The user has also the choice to calculated LODs without using fully uninformative markers, i.e. markers with homozygous and identical genotypes in all tested individuals. However, removing the uninformative markers can lead to a substantial information loss and is therefore not recommended. Allegro should not be limited in the number of markers; however the pedigrees have to be of a moderate size. With regards to large scale SNP data and multipoint analyses an important issue has to be mentioned here: Although the authors of Allegro claim that Allegro is not limited in the number of markers Allegro can run into severe problems in assigning correct haplotypes. The user would not notice. It is possible to analyze 500 markers on a chromosome in a piece. We experienced a number of situations where the LOD score dropped significantly when using many markers, sometimes from 3 to 0. The reasons for that are not yet clear to us. Most likely it is not due to the marker number but to the used allele frequencies. Haplotype assignment problems can occur when a rare allele is made to a common or a common to a rare allele. Even population reference allele frequencies must be handled with care. However, it cannot be excluded beyond reasonable doubt that the marker number itself or limitations of the Lander-Green algorithm might play a role. To circumvent the problem we implemented the possibility to analyze sets of markers and/or predefined chromosomal cm intervals. Here, we propose the following guidelines when dealing with large-scale SNP data: 1. Try to avoid SNPs that are very close to each other. Allegro, GeneHunter, Merlin and other programs assume linkage equilibrium between markers. That rule can be easily broken when using many markers in close proximity. However, if such a situation occurs, easylinkage will set the distance between such markers to cm. 2. When analyzing an entire chromosome or even the whole genome perform more than just one analysis: 6

7 a. Analysis of entire chromosomes b. Analysis of blocks of maximal 100 markers c. Analysis of blocks with a smaller marker number in order to overlap regions of b. d. Analysis of the region where you observe peaks without using blocks of markers e. Analysis of the region where you observe peaks using blocks of markers f. Use different allele frequency algorithms (preferentially codominant (equal allele distribution) when dealing with inbred or larger families) 3. Analyze your data with and without fully-uninformative markers, i.e. markers with homozygous, 1-allelic genotypes for every genotyped subject. Homozygous markers genotypes can add significant information to the results of multi-point analyses. They can be left aside in two-point linkage analyses. easylinkage does provide many error checking routines and many other options that make the program really user-friendly. The user gets software in hand which was extensively tested by the developers. The programmers themselves are tied up in linkage projects as well, therefore always programming very close to the everyday needs. Many users noted the limited pedigree drawing abilities of all programs. Only GeneHunter provides pedigree plots, however they are very limited. Therefore, easylinkage extends the GeneHunter plots by used markers with their genetic position and in addition, it provides input files for the software HaploPainter. This program draws very nice pedigrees including a colored presentation of markers, position, and haplotypes plus recombination events. We have realized that some scientists are not used to the rather complicated setup of the PERL interpreter and all the additional modules easylinkage needs to run correctly. Therefore, we compiled the program into a Windows binary and packed all the necessary files into a user friendly setup system. The use of the program is free of charge. We appreciate any comment or bug report. We would also appreciate to register your with us in order to keep you updated with bug fixes, newer versions and stuff like that. Good luck and have fun! If used, please cite the program for STRP analyses with: Lindner TH, Hoffmann K: easylinkage: A PERL script for easy and automated two- /multi-point linkage analyses. Bioinformatics 21: , 2005 and for SNP analyses with: Hoffmann K, Lindner TH: easylinkage Plus Automated linkage analyses using largescale SNP data. Bioinformatics 21: ,

8 II. Specific options / limitations of the covered linkage programs 1. FastLink: It is recommended to keep the Recode Alleles option activated. FastLink cannot analyze more than one family with loops. That severe bug has not been fixed for years now. However, this situation can be handled at best by SuperLink. 2. SuperLink: If you use pedigrees in your analysis whose ID is an integer you must number them continuously starting with 1. Otherwise the program will hang up completely. Therefore, it is recommended not to use integers but alphanumeric IDs as family identifiers. 3. SPLink: The program cannot analyze X markers (internal bug). 4. GeneHunter: It is recommended to keep the Recode Alleles option activated. Gene- Hunter Plus cannot perform X analyses. However, if the X chromosome is chosen X will be automatically analyzed using GeneHunter. GeneHunter is limited in marker number. You have to be careful with the haplotype construction algorithm. If you have more than 100 markers on one chromosome you will get a warning followed by a set analysis with 100 markers max for each set. This procedure enables the analyses of thousands of markers. However, you have to run your analyses at least twice with different set sizes in order not to miss the linkage signal. 5. GeneHunter TwoLocus: In order to run this program correctly you MUST copy the provided program touch.exe (in the GeneHunter directory) into the Windows\system32 directory. Be aware that you must provide an inheritance model for this analysis. Since providing the true underlying inheritance model would be a game in case of complex traits, we do not recommend this procedure for those diseases. In addition the program tests different inheritance models automatically. The resulting p values or LODs will not be corrected for multiple testing. The program might be useful however under strong consideration of its potential pitfalls. Analyses with chromosome X are not supported yet. 6. GeneHunter Imprinting: Again imprinting models will only affect the computing of parametric LODs but will have no effect on non-parametric analyses. 7. Allegro: The use of the LIN and EXP models results sometimes in information scores >1. This bug has not been fixed yet by the authors. If you use more than 100 markers a set analyses as we designed it for GeneHunter is recommended. If you want to simulate multipoint data you cannot enter smaller cm distances than 0.5 cm even if you state UNITS centimorgans explicitly. There is probably a bug in the later option for the OPT file. 8. Merlin: The HWE analysis option as well as the NON-Mendelian error check routine was implemented. Note, that the HWE routine disregards any pedigree structure and does just perform χ 2 significance tests but NOT exact HWE tests (Fisher). That limits the use of that program. 9. SimWalk: The program cannot work with cm distances. easylinkage does therefore a transformation of cm map positions into recombination fractions by the use of the Haldane mapping function. The user will not recognize that. For the time being it is not possible to analyze microsatellite markers together with SNPs in one finemapping project. That is due to the limited max marker capacity of SimWalk (n = 63). 10. SLink: The program can now analyze families with loops. Loops will be broken by the unknown l option from the FastLink package (A. Schaffer). 11. PedCheck: No specific notes. 8

9 III. Installation Starting with version 3.0 we decided to provide a precompiled version in a setup routine making it rather convenient to start with. Start the setup program easylinkage_v5.05_setup.exe and follow the instructions. IMPORTANT! Several subdirectories will be created. Runtimes for Allegro, PedCheck, GeneHunter Imprinting, GeneHunter TwoLocus, SimWalk, PedCheck cannot be provided. See the introductory notes regarding this issue. Once the installation is complete you can use the program right away. easylinkage requires an INI file easylinkage_setup.ini without that it would not run at all. The INI file can be edited manually. The options are self explaining. In brief, you can predefine penetrances, models, the path of the linkage runtimes and other parameters as the default setup for easylinkage. Here is an example of a valid INI file (that might need modification depending on your installation paths). Any comment line starts with! where you can find the options that can be used for the subsequent option lines:! Whenever a line starts with an "!" this lane will be disregarded. Any option! that can be changed will be commended with "!" at the beginning of a a lane. [General options]! The default marker locus analysis. Options:! 1! 2 9

10 LOCUS_TYPE=1! The path of the main program. PROGRAM=D:\SoftTestWare\linkage_v5.05.exe! The project type. Options:! Microsatellites! SNPs PROJECT=Microsatellites! The path were the maps reside. MAP_DIR=D:\SoftTestWare\Maps! The default map. Options:! Marshfield STRP map! decode STRP map! LDB STRP map! SLM1 SNP map! Marshfield SNP map! decode SNP map! other user-defined maps MAP=Marshfield STRP map! Chromosomal interval defaults in cm. CHR_FROM_cM=0.00 CHR_TO_cM= ! Most programs work better if alleles will be provided as continuous values! starting fromm 0. This options enables this procedure. Options:! On! Off RECODE_ALLELES=On! Some programs have limits in terms of handling long identifiers for either! family or subject IDs. Options:! On! Off RECODE_IDs=On! Most analyses should be run with sex-averaged map data. Options:! Sex averaged! Female! Male GENDER_MAP=Sex averaged! The marker name delimiter is important since it ensures the correct recognition! of marker files for microsatellite data. The default is "\_". Note, that only! the "_" part is the delimiter. Therefore, if you want to use "-" instead, enter! "\-". MARKER_NAME_DELIMITER=\_! The default marker file extension. Again, only important for microsatellites. MARKER_FILE_EXT=abi! The following three lines tell easylinkage where to find subject IDs, and! alleles. If your format differs from the default, you may enter your own! configuration here. SUBJECT_ID_COL=3 ALLELE_1_COL=4 ALLELE_2_COL=5! PedCheck defaults. Please do not switch from "Off" to "On". Might cause problems. PED_CHECK=Off! Default PedCheck path. If the entry is wrong all PedCheck options will be! deactivated. PEDCHECK=D:\SoftTestWare\PedCheck\pedcheck_win.exe! Tables in final ouput window. This option is not really neccessary since it! will have no effect on text outputs the program will generate. Options:! On! Off TABLES=Off! Sounds defaults. Options:! On! Off SOUNDS=Off! LOGging defaults. Options:! 1... On! 0... Off LOGGING=1 [FastLink]! Description of program is it is desired to appear in the MainScreen of easylinkage. FASTLINK_DESC=FastLink v4.1 - Two-Point Parametric Linkage Analysis! Paths of linkage auxilliary programs. MAKEPED_FL=D:\SoftTestWare\FastLink\MakePED.exe UNKNOWN_FL=D:\SoftTestWare\FastLink\Unknown.exe MLINK_NO_LOOP_FL=D:\SoftTestWare\FastLink\MLink_noloop.exe MLINK_LOOP_FL=D:\SoftTestWare\FastLink\MLink_loop.exe! Only required for pedigrees with loop(s). Options: 10

11 ! On! Off LOOP=Off! Default inheritance model. Options:! Dominant! Recessive INHERITANCE_FL=Dominant! Default number of liability classe. Please do not change!!! LIABILITY_FL=1! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_ALGORITHM_FL=All individuals from marker file! Default frequency of the disease allele. DISEASE_FREQ_MT_FL=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_FL=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_FL=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_FL=0.0000! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_FL=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_FL=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_FL=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_FL=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_FL=0.0000! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_FL=Only "TOTALS" plot! Default recombination increment. RECOMBINATION_INCREMENT_FL=0.05! Default recombination finishing value. RECOMBINATION_FINISHING_VALUE_FL=0.45 [SuperLink]! Description of program is it is desired to appear in the MainScreen of easylinkage. SUPERLINK_DESC=SuperLink v1.6 - Two-Point Parametric Linkage Analysis! Default paths for SuperLink programs. MAKEPED_SUP=D:\SoftTestWare\FastLink\MakePED.exe UNKNOWN_SUP=D:\SoftTestWare\SuperLink\Unknown.exe SUPERLINK_PROG=D:\SoftTestWare\SuperLink\SuperLink_v1.6.exe! Default inheritance model. Options:! Dominant! Recessive INHERITANCE_SUP=Dominant! Default number of liability classe. Please do not change!!! LIABILITY_SUP=1! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_ALGORITHM_SUP=All individuals from marker file! Default frequency of the disease allele. DISEASE_FREQ_MT_SUP=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_SUP=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_SUP=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_SUP=

12 ! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_SUP=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_SUP=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_SUP=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_SUP=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_SUP=0.0000! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_SUP=Only "TOTALS" plot! Default recombination increment. RECOMBINATION_INCREMENT_SUP=0.05! Default recombination finishing value. RECOMBINATION_FINISHING_VALUE_SUP=0.45 [SPLink]! Description of program is it is desired to appear in the MainScreen of easylinkage. SPLINK_DESC=SPLink v Two-Point Nonparametric Linkage Analysis (Affected Sib Pairs)! Default Path for SPLink program. SPLINK=D:\SoftTestWare\SPLink\SPLink_v1.09.exe! Only include families with, at least, one affected sib pair. Switch this! option OFF if you want such families to contribute\nto estimation of the! haplotype frequencies. AT_LEAST_ONE_ASP=1! Only include "complete" families in the analysis (i.e., families with! both parents typed). ONLY_INCLUDE_COMPLETE_FAMILIES=0! Allow multiple nuclear families from one pedigree (although the relationship! between these families will be ignored). If not set, only the first nuclear! family encountered in each pedigree is used. ALLOW_MULT_NUC_FAM_FROM_ONE_PED=1! Maximum number of nuclear families. MAX_NO_NUC_FAM=1000! Maximum number of persons on data file. MAX_NO_PERS=10000! Number of marker loci. MARKER_LOCI_NO=1! Score tests. Options:! 2 df test! 1~2 df "possible triangle" test! 1 df test with the constraint z1 = 0.5! 1 df test with the constraint z1 = 2 * z0 SCORE_TEST=2 df test! Weight multiple sibpair pair comparisons within the same nuclear family by! 2/A, where A is the number of affected sibs. WEIGHT_MULT_ASP=1! Variance estimator. Options:! Naive! Theoretical! Empirical VARIANCE_ESTIMATOR=Naive! Bootstrap significance test. Options:! 0! 1 BOOTSTRAP=0! Number of bootstrap samples. BOOTSTRAP_SAMPLES=0! Maximum allowable ambiguity for parental haplotyping. MAX_ALLOWABLE_AMBIGUITY=500! Amount of output. Please do not change this option. OUTPUT_AMOUNT=Minimum output [GeneHunter]! Description of program is it is desired to appear in the MainScreen of easylinkage. GENEHUNTER_DESC=GeneHunter - Multipoint Linkage Analysis (GH, GHP, GH Imprinting)! GeneHunter paths GENEHUNTER_AUTOSOMES=D:\SoftTestWare\GeneHunter\gh2.1r5_mw.exe GENEHUNTER_X=D:\SoftTestWare\GeneHunter\xghp1.2_mw.exe! GeneHunter Plus paths GENEHUNTER_PLUS_AUTOSOMES=D:\SoftTestWare\GeneHunter\ghp1.2_mw.exe GENEHUNTER_PLUS_X=D:\SoftTestWare\GeneHunter\xghp1.2_mw.exe! ASM path (only required for Genehunter Plus). 12

13 ASM=D:\SoftTestWare\GeneHunter\asm.exe! GeneHunter Imprinting paths. GENEHUNTER_IMPRINTING_AUTOSOMES=D:\SoftTestWare\GeneHunter\ghi2.1r3beta_mw.exe GENEHUNTER_IMPRINTING_X=D:\SoftTestWare\GeneHunter\xghi1.3_mw.exe! GeneHunter TwoLocus paths. GENEHUNTER_TWOLOCUS=D:\SoftTestWare\GeneHunter\ghtl1.3_mw.exe GENEHUNTER_TWOLOCUS_GNUPLOT=D:\SoftTestWare\GnuPlot\wgnuplot.exe! GeneHunter TwoLocus options.! Disease frequency at locus 1 DISEASE_FREQ_1=0.001! Disease frequency at locus 2 DISEASE_FREQ_2=0.001! Locus 1 chromosome. LOCUS_1_CHR=1! Locus 2 chromosome. LOCUS_2_CHR=2! cm interval at locus 1. LOCUS_1_cM_FROM=0.00 LOCUS_1_cM_TO= ! cm interval at locus 1. LOCUS_2_cM_FROM=0.00 LOCUS_2_cM_TO= ! 4x4 penetrance matrix. L1 = Locus 1, L2 = Locus 2, WW = Wt/Wt, MM = Mt/Mt PEN_L1_WW_L2_WW= PEN_L1_WW_L2_MW= PEN_L1_WW_L2_WM= PEN_L1_WW_L2_MM= PEN_L1_MW_L2_WW= PEN_L1_MW_L2_MW= PEN_L1_MW_L2_WM= PEN_L1_MW_L2_MM= PEN_L1_WM_L2_WW= PEN_L1_WM_L2_MW= PEN_L1_WM_L2_WM= PEN_L1_WM_L2_MM= PEN_L1_MM_L2_WW= PEN_L1_MM_L2_MW= PEN_L1_MM_L2_WM= PEN_L1_MM_L2_MM= INTERVAL_ANALYSIS_GH=1! Number of markers per marker set. Please do not use numbers >100! INTERVAL_MARKER_NO_GH=100! Inheritance model. Only in effect for parametric analyses. Options:! Dominant! Recessive INHERITANCE_GH=Dominant! Number of liability classes. Please do not change! LIABILITY_GH=1! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_ALGORITHM_GH=All individuals from marker file! Frequency of the disease allele. DISEASE_FREQ_MT_GH=0.0010! Number of intermarker anlysis points. Must be an integer >0. STEPS_GH1=5 STEPS_GH2=5! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_GH=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_GH=1.0000! Default autosomal penetrance for mt/wt genotype. Only in effect for! imprinting analyses. PENETRANCE_MT_WT_GH=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_GH=0.0000! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_GH=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_GH=0.9900! Default X chromosomal (female) penetrance for mt/wt genotype. Only! in effect for imprinting analyses. 13

14 PENETRANCE_MT_WT_FEMALE_GH=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_GH=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_GH=0.9900! Default X chromosomal (male) penetrance for mt/wt genotype. Only! in effect for imprinting analyses. PENETRANCE_MT_WT_MALE_GH=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_GH=0.0000! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_GH=Only "TOTALS" plot! Default GeneHunter programs. Note, those options will only be available! within the "GeneHunter options" menu if those programs were installed!! GeneHunter v2.1r5! GeneHunter PLUS v1.2 - ASM Lin OFF - ASM Exp OFF! GeneHunter PLUS v1.2 - ASM Lin OFF - ASM Exp ON! GeneHunter PLUS v1.2 - ASM Lin ON - ASM Exp OFF! GeneHunter PLUS v1.2 - ASM Lin ON - ASM Exp ON! GeneHunter Imprinting OFF / MODscore ON! GeneHunter Imprinting ON / MODscore OFF! GeneHunter Imprinting ON / MODscore ON PROGS_GH=GeneHunter v2.1r5! Default haplotyping algorithm. Options:! Off! Viterbi! MaxProb HAPLOTYPING_GH=Off! Count recombination events. This procedure is time-consuming. Options:! On! Off COUNT_RECS_GH=On! Eliminating less informative subjects. Options:! On! Off DISCARD_GH=Off! Defines the maximum allowable BIT for GeneHunter calculations. Options:! Any number <24 makes sense otherwise the program might do funny things! MAX_BITS_GH=19 [Allegro]! Description of program that should appear in the MainScreen of easylinkage.! ALLEGRO_DESC=Allegro v2.0f - Two-/MultiPoint Para-/NonParametric Linkage Analysis ALLEGRO_DESC=Allegro v1.2c - Two-/MultiPoint Para-/NonParametric Linkage Analysis! Default Allegro path.! ALLEGRO_EXE=D:\SoftTestWare\Allegro\allegro_v2.0f.exe ALLEGRO_EXE=D:\SoftTestWare\Allegro\allegro_v1.2c.exe! Decide whether single-point analysis should be activated as default. Option:! On! Off ALLEGRO_SINGLE_POINT=On! Decide whether multi-point analysis should be activated as default. Option:! On! Off ALLEGRO_MULTI_POINT=On! Decide whether parametric analysis should be activated as default. Option:! On! Off ALLEGRO_PARAMETRIC=Off! Decide whether nonparametric analysis should be activated as default. Option:! On! Off ALLEGRO_NONPARAMETRIC=On! Default activation for LIN model analyses. Only in effect when nonparametric! analyses were switched on. ASM_LIN_AL=Off! Default activation for EXP model analyses. Only in effect when nonparametric! analyses were switched on. ASM_EXP_AL=On! Interval analysis. 1=On, 0=Off. Options:! 1! 0 INTERVAL_ANALYSIS_AL=0! Number of markers per marker set. In contrast to GeneHunter there is no limit! in Allegro. 14

15 INTERVAL_MARKER_NO_AL=100! Inheritance model. Only in effect for parametric analyses. Options:! Dominant! Recessive INHERITANCE_AL=Dominant! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_ALGORITHM_AL=All individuals from marker file! Number of liability classes. Please do not change! LIABILITY_AL=1! Frequency of the disease allele. DISEASE_FREQ_AL=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_AL=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_AL=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_AL=0.0000! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_AL=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_AL=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_AL=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_AL=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_AL=0.0000! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_AL=Only "TOTALS" plot! Default scoring function. options:! All! Pairs! HOMOZ! ROBDom! MNAllele SCORING_FUNCTION_AL=Pairs! Default weighting function. If using the "File with family weights" option! you must provide appropriate weighting files. Options:! Equal! File with family weights! Power:0.1! Power:0.2! Power:0.3! Power:0.4! Power:0.5! Power:0.6! Power:0.7! Power:0.8! Power:0.9! Power:1.0 WEIGHTING_AL=Equal! Only in effecte when using "File with family weights" option. WEIGHT_FILE_AL=<NONE>! Analysis steps between two markers STEPS_AL=5! Write uninformative subjects to a file. Options:! On! Off UNINFORMATIVE_AL=Off! Maximum RAM to be allocated. Here 1000 = 1GB. MAXMEMORY_AL=1000! Sexspecific analysis. Options:! On! Off SEXSPECIFIC_AL=Off! Default haplotyping algorithm. Options:! On! Off 15

16 HAPLOTYPING_AL=Off [Merlin]! Description of program is it is desired to appear in the MainScreen of easylinkage. MERLIN_DESC=Merlin v Multipoint Nonparametric Linkage Analysis! Default path for Merlin (autsomal program) MERLIN_EXE=D:\SoftTestWare\Merlin\Merlin_v1.0.1.exe! Default path for Merlin (X chromosomal program) MERLINX_EXE=D:\SoftTestWare\Merlin\MerlinX_v1.0.1.exe! Default path for PedStats. Required for HWE check and NON-Mendelian tests. PEDSTATS_EXE=D:\SoftTestWare\Merlin\PedStats_v0.6.3.exe! Interval analysis. 1=On, 0=Off. Options:! 1! 0 INTERVAL_ANALYSIS_M=0! Number of markers per marker set. Please do not use numbers >100! INTERVAL_MARKER_NO_M=100! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_MGORITHM_M=All individuals from marker file! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_M=Only "TOTALS" plot! Analysis steps between two markers. Must be integer >0. STEPS_M=5! Default haplotyping algorithm. Options:! Off! Best! Sample! All HAPLOTYPING_M=Off! Default IBD calculations. Options:! 1 = On! 0 = Off IBD_M=0! Default kinship analyses. Options:! 1 = On! 0 = Off KINSHIP_M=0! Default information score analyses. Options:! 1 = On! 0 = Off LIKELIHOOD_M=0! Default scoring function. Options:! All! Pairs! All AND Pairs SCORING_FUNCTION_M=All! Maximum bits allowed (bits) BITS_M=24! Maximum RAM to be allocated (megabytes). MEMORY_M=1000! Maximum time to be allowed (minutes). MINUTES_M=1000! Decide whether parametric analysis should be activated as default. Option:! On! Off MERLIN_PARAMETRIC=Off! Default activation for LIN model (nonparametric) analyses. ASM_LIN_M=On! Default activation for EXP model (nonparametric) analyses. ASM_EXP_M=Off! Inheritance model. Only in effect for parametric analyses. Options:! Dominant! Recessive INHERITANCE_M=Dominant! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only 16

17 ! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian! Frequency of the disease allele. DISEASE_FREQ_M=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_M=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_M=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_M=0.0000! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_M=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_M=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_M=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_M=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_M=0.0000! LD removal algorithm.! 1 = On! 0 = Off LD_REMOVAL=0! LD removal method.! r... By r squared! d... By distance clusters (see Merlin manual) LD_METHOD=d! Distance in cm for the "Distance clusters method". LD_DISTANCE=0.1! R squared for the "r squared method" LD_R_SQUARED=0.1 [SimWalk]! Description of program is it is desired to appear in the MainScreen of easylinkage. SIMWALK_DESC=SimWalk v Multipoint Linkage Analysis! Default path for SimWalk (general program). SIMWALK_EXE=D:\SoftTestWare\SimWalk\SimWalk_v2.91.exe! Default path for SimWalk (SNP program). SIMWALK_SNP_EXE=D:\SoftTestWare\SimWalk\SimWalkSNP_v2.91.exe! Default plot option. Options:! Plots for all families! Only "TOTALS" plot DESIRED_PLOT_SW=Only "TOTALS" plot! Default haplotyping algorithm. Options:! On! Off HAPLOTYPING_SW=Off! Default allele frequency algorithm. Options:! Codominant! All individuals from marker file! All individuals from pedigree file! Founders only! First subject of an ASP! AFFYMETRIX Reference - Asian! AFFYMETRIX Reference - African American! AFFYMETRIX Reference - Caucasian ALLELE_FREQ_ALGORITHM_SW=All individuals from marker file! Interval analysis. 1=On, 0=Off. Options:! 1! 0 INTERVAL_ANALYSIS_SW=0! Number of markers per marker set. If using the general SimWalk! program (no SNPs) this value must be integer <64. INTERVAL_MARKER_NO_SW=63! Default inheritance model for parametric analyses. Options:! Dominant! Recessive INHERITANCE_SW=Dominant! Number of liability class. Please do not change!!! LIABILITY_SW=1! Frequency of the disease allele. DISEASE_FREQ_MT_SW=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_SW=

18 ! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_SW=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_SW=0.0000! Default X chromosomal (female) penetrance for mt/mt genotype. PENETRANCE_MT_MT_FEMALE_SW=0.9900! Default X chromosomal (female) penetrance for wt/mt genotype. PENETRANCE_WT_MT_FEMALE_SW=0.9900! Default X chromosomal (female) penetrance for wt/wt genotype. PENETRANCE_WT_WT_FEMALE_SW=0.0000! Default X chromosomal (male) penetrance for wt/mt genotype. PENETRANCE_WT_MT_MALE_SW=0.9900! Default X chromosomal (male) penetrance for wt/wt genotype. PENETRANCE_WT_WT_MALE_SW=0.0000! Sampling (simulation) analyses. 1=On, 0=Off. Options:! 1! 0 SAMPLING_SW=0! Default haplotyping. Options:! On! Off HAPLOTYPING_SW=Off! Parametric analyses. 1=On, 0=Off. Options:! 1! 0 PARAMETRIC_SW=1! Nonparametric analyses. 1=On, 0=Off. Options:! 1! 0 NONPARAMETRIC_SW=1! IBD analyses. 1=On, 0=Off. Options:! 1! 0 IBD_SW=0! Mistyping analyses. 1=On, 0=Off. Options:! 1! 0 MISTYPING_SW=0! Model heterogeneity. Alpha=1.00 if no heterogeneity. ALPHA_SW=1.00! Number of replicates for simulation analyses. REPLICATES_SW=10 [SLink]! Description of program is it is desired to appear in the MainScreen of easylinkage. FASTSLINK_DESC=FastSLink v Two-Point Pedigree Simulation Analysis! Default SLink paths. MAKEPED_SL=D:\SoftTestWare\FastSLink\MakePED.exe UNKNOWN_SL=D:\SoftTestWare\FastSLink\Unknown.exe SLINK_SL=D:\SoftTestWare\FastSLink\FastSLink.exe MSIM_SL=D:\SoftTestWare\FastSLink\MSim.exe! Default inheritance model for parametric analyses. Options:! Dominant! Recessive INHERITANCE_SL=Dominant! Number of liability classes. Please do not change!!! LIABILITY_SL=1! Frequency of the disease allele. DISEASE_FREQ_MT_SL=0.0010! Default autosomal penetrance for mt/mt genotype. PENETRANCE_MT_MT_SL=1.0000! Default autosomal penetrance for wt/mt genotype. PENETRANCE_WT_MT_SL=1.0000! Default autosomal penetrance for wt/wt genotype. PENETRANCE_WT_WT_SL=0.0000! Default number of marker alleles. MARKER_ALLELE_NO=4! Default number of recombinations. REPLICATIONS_SL=1000! Default recombination starting value. REC_VALUE_SL= ! Default recombination increment. REC_INCREMENT_SL= ! Default recombination finishing value. REC_FIN_SL=

19 Important: If you have previously used another version of easylinkage please delete last_used_entries.txt from the directory where easylinkage resides. IV. Input files IV.1 Microsatellite marker projects Marker files (contain genotypes) Here, genotypes have to be provided in separate files. In a project with 422 microsatellite markers you need therefore 422 appropriate files. Those files have the following structure: Example D1S1609_1_022.abi : MARKER LANE ID A_1 A_2 D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ D1S KB_0202_0201_ easylinkage uses only columns 1, 3, 4, 5. All other columns are optional. Column 2 is required, however, the column content does not matter at all. If this required information is in another place within your files you must edit the INI file appropriately (positions: SUB- JECT_ID_COL=, ALLELE_1_COL=, ALLELE_2_COL= ) and enter the correct columns. Lanes that start with # or - will be ignored during the run. Blanks must be depicted as or 0 0. The file extension can also be changed using in the INI file (position: MARKER_FILE_EXT= ). Marker delimiter is _ in the presented example (position: MARKER_NAME_DELIMITER=\...). PEDFILE (pedigree structure file in LINKAGE format) This file is required in general LINKAGE format. Person IDs have to match exactly to those in the marker files. Person / family IDs MUST be unique throughout the pedigree information file! Otherwise the files cannot be assembled appropriately prior any linkage analysis. Example p_gsdm_t2dm.pro : D_0000_7059 D_0000_7059_ D_0000_7059 D_0000_7059_ D_0000_7059 D_0000_7059_0201 D_0000_7059_0101 D_0000_7059_ D_0000_7059 D_0000_7059_0202 D_0000_7059_0101 D_0000_7059_ D_0000_7059 D_0000_7059_0203 D_0000_7059_0101 D_0000_7059_ D_0000_7059 D_0000_7059_0204 D_0000_7059_0101 D_0000_7059_ D_0000_7059 D_0000_7059_0205 D_0000_7059_0101 D_0000_7059_ D_0000_7059 D_0000_7059_0206 D_0000_7059_0101 D_0000_7059_ D_0000_7159 D_0000_7159_ D_0000_7159 D_0000_7159_

20 D_0000_7159 D_0000_7159_0201 D_0000_7159_0101 D_0000_7159_ D_0000_7159 D_0000_7159_0202 D_0000_7159_0101 D_0000_7159_ D_0000_7159 D_0000_7159_0203 D_0000_7159_0101 D_0000_7159_ D_0000_7159 D_0000_7159_0204 D_0000_7159_0101 D_0000_7159_ D_0000_7159 D_0000_7159_0205 D_0000_7159_0101 D_0000_7159_ D_0000_7159 D_0000_7159_0206 D_0000_7159_0101 D_0000_7159_ Column 1: Column 2: Column 3: Column 4: Column 5: Column 6: Optionally: Column 7: Column 8: Family ID Person ID Father ID Mother ID Sex (1 = male, 2 = female, 0 = unknown) Affection status (1 = unaffected, 2 = affected, 0 = unknown) DNA availability (2 = available, 0 = not available). This column is only required for SLink exclusively. Must be provided if you use more than 1 liability class. This is the liability class column. IV.2 SNP projects The pedigree structure file is identical to those used for microsatellite marker projects. Only the marker files are different. Here, all markers and genotypes have to be in a single file. The format is as follows: SNP_ID SNP_A BB BB BB BB BB BB BB BB AA BB BB BB AB SNP_A AA AB AB AA 00 AB AA AB AB AB 00 AB AB SNP_A AA AA AA AA AA AA AA AA AA AA AA AA AA SNP_A AA AA AA AA AA AA AA AA AA AA AA AA AA SNP_A BB AB AB AB 00 BB AB AB 00 BB BB SNP_A AA AA AA AA BB AA AA AB AB AA AA AB 00 SNP_A BB AB BB AB BB BB BB BB BB BB BB BB BB SNP_A AB AB AA AA BB AA BB AB AB AB AB AB AB SNP_A BB AA AB AB AB AB AB AB BB BB AB AB AA SNP_A BB BB BB BB BB BB BB BB BB BB BB BB BB SNP_A AB AB AB AB BB AB AB AB BB AB BB AB BB SNP_A AA AA AA AA AB AA AA AA AA AB AA AA AA SNP_A AB AA AA AA AA AA AA AB BB AA AA AA AA SNP_A BB AB BB AB BB AB BB AB BB BB AB AB BB SNP_A BB BB BB BB BB BB BB BB BB BB BB BB BB SNP_A AB AA AB AA AB AA AA AB AB AA AB AA AB SNP_A AA AB AA AB BB AB AA AA AB AB AA BB AA SNP_A AB AA AB AB BB AA AA BB AA AA AA AB AB SNP_A AA AB AA AA BB AB AA AA AB BB AB AB AB SNP_A AB AB AA AB AB AA AB BB AB AB AA AB BB SNP_A AA BB AB AB AA AB AB BB AB AB BB AB AB SNP_A AB AA AB AA AA AA AB AA AA AB AB AA AB SNP_A AA AB AA AA AA AA AB AA AA AA AA AA AA SNP_A AA AA AA AA AA AA AA AA AB AB AB AA AA SNP_A AB BB BB BB AB BB BB AB AB BB AB AB AA SNP_A AA AB AA AB AA AB AA AB BB AB AB AA AB SNP_A AA AB AA AA AB AB AB AB AB AB AA AB AB SNP_A AB AB AB AB AB AB AA AA AB BB BB BB SNP_A AB AB AB AA AB AB AA AB AB AB AA AA SNP_A AA AA AA AA AA AA AA AA BB AA AB AA AB The first line contains the person IDs. On the left hand-side the SNPs are listed. IDs of SNPs can be those from Affymetrix, dbsnp, or from The SNP consortium (TSC) (blue). Alleles must be depicted by A or B, blanks as 00 (bold green). Alternatively, genotypes of SNPs and microsatellite markers can be put in one file. In that case, genotypes must be numbers with the individual alleles separated by an (example:

21 or ). The merge_snp program can merge SNP files with individual marker genotypes files. IV.3 Liability classes definition files Liability classes files will be recognized by easylinkage by the file extension liab. You MUST stick to the structure as shown in the following example. There is no limit in the use of liability classes. You can also provide as many liability classes files as you want. Example Two_classes.liab : 2 << NO. OF LIABILITY CLASSES # Autosomes << PENETRANCES # X chromosome << PENETRANCES For GeneHunter TwoLocus a 4x4 penetrance matrix must be provided for each liability class (X analyses are not possible): 2 << NO. OF LIABILITY CLASSES # Autosomes << PENETRANCES << PENETRANCES For further details, please refer to the GeneHunter TwoLocus manual. IV.4 Weight files (only for Allegro; conditional linkage analyses) Weight files can be generated with easylinkage. For that you need a results file of a nonparametric Allegro analysis. To generate weight files please go to Allegro options, click on the button WF, select an Allegro results file. Then, for each marker where the TOTALS analysis generated a NPL score>0 weight files will be generated. There will be three different weight files: 1. Weight 1-0 (families with NPL 0 => weight 0, families with NPL<0 => weight 1) 2. Weight 0-1 (families with NPL 0 => weight 0, families with NPL>0 => weight 1) 3. Weight prop (families with NPL<0 => weight 0, families with NPL>0 => weight=npl) 21

22 The generated weight files will be stored under the subdirectory Weighting_files within the folder you set in the main screen of easylinkage. For subsequent analyses those files will be available in the File: drop-down menu. IV.5 Marker map files From version 5.00 on easylinkage can make use of user defined marker maps. That means the user can generate as many marker maps as desired. However, those map files must contain specific headers enabling the program to differentiate between microsatellite and SNP marker maps. By the way, microsatellite marker maps can contain SNPs for finemapping purposes as well. We established the difference between microsatellite marker and SNP projects since marker genotypes will be provided in single marker files for microstellite marker projects and in just one file containing all genotypes for SNP projects. A marker map file header must look like the following example for microsatellite marker projects: # Map name: decode STRP map # Projects: STRP #_MARKER LOCUS cm_ave cm_female cm_male CHR PHYS_POS D1S243 AFM214YG7 01: : : Of this header just those lines starting with # are important: 1. line: Description of marker map. Please do not change the first part ( # Map name: ) which is absolutely essential for correct functioning! 2. line: Assign your map to either SNP or STRP (microsatellite marker) projects. Please do not change the first part ( # Projects: ) which is absolutely essential for correct functioning! 3. line: Of this line only cm_ave or cm_female or cm_male are important. For STRP maps cm_ave must be the 3. term in the line (as in the above example), cm_female the 4. term, and cm_male the 5. term. For SNP projects cm_ave must be the 2. term and the others the subsequent 3./4. terms. If any of those terms is not found in this line just the remaining position columns will be considered for choosing gender maps in the main screen of the program. Note: The #_ at the beginning must stay this way! This structure enables to add user-defined marker maps. Remember, if a file having the extension.map does not contain this header this particular file will not be recognized as a correct marker map file. On the other side, try to avoid identical marker map descriptions in different maps since this description will be displayed in the main screen of the program. So having identical descriptions will not allow differentiating the content of each of those maps for subsequent analyses. IV.6 QTL analysis files (only for Merlin) A QTL file contains just 2 columns: Column 1: Subject ID Column 2: Quantitative value of a parameter (at the time only one parameter per file is allowed 22

23 The file MUST end with.qtl in order to be recognized by easylinkage. Here is an example: Example T2D_triglycerides.qtl :

24 V. Running the program - Welcome Screen Before the MainScreen opens easylinkage checks the availability of linkage programs. Note, that the program executables might be installed but will be given as not existing by the error message screen. Then you should check the easylinkage_setup.ini file whether the paths for the executables are correct. Otherwise, a similar screen like the following will appear: If the tests were finally ok the MainScreen comes up: 24

25 Chromosomes can be chosen individually up to an entire genome scan. If some programs do not use all options of the main screen, those options will be deactivated. By clicking the DIR button the directory that contains the ABI files and the PEDFILE can be chosen. By the way, all result files will be moved into a new subdirectory that contains a time stamp. In this way, older analyses cannot be overwritten. Alternatively, the target directory can be entered manually or pasted from the explorer window into the entry window to the right of the DIR button. After choosing the working directory the program immediately scans this directory for PEDFILES. By clicking Program options the user can set almost all options the different linkage programs provide. Note: If you need help just let the cursor rest over any button or checkboxes for a couple of milliseconds. A balloon help will appear. Options: Recode alleles Recode IDs Mendelian test NON-Mendelian test This will lead to a continuous recoding of all alleles within your marker files (originals will not be changed but only the input files which are generated by easylinkage). Some programs require this procedure, otherwise they will not run. You can apply this option to any program. Only Merlin does not depend on this routine. If your original allele numbers become larger than a certain integer Allegro will not run as well. In most cases it is recommended to leave this option activated. Family and subject IDs will recoded to integers. At the end of the run the recoded IDs will be transferred to their original state. This procedure is sometimes required. For example, GeneHunter has problems with certain ID structure. SimWalk allows only 8 letters for IDs and marker names. In case of SimWalk this option will be automatically activated if such a situation will be detected by easylinkage. As it is the case for the Recode alleles option family and subject IDs will be reset to their original state at the end of the run. This options will initiate a PedCheck run (if PedCheck was installed on your system). PedCheck tests all your marker files for Mendelian errors. If Mendelian errors were detected you will have the chance to exclude the affected marker from subsequent linkage analyses. This will initiate a Merlin test routine which can detect genotyping errors within a haplotype. For instance, if PedCheck detects no Mendelian errors for an individual marker, the same marker could generate a double recombination in a haplotype. This kind of error will be detected by this routine. In general, it is recommended to leave this option switched on. 25

26 However, you will depend on a correct haplotype assignment by the internal Merlin routines. The correctness can depend on different factors such as the use of a particular allele frequency algorithm. HWE test Sounds Tables Redirect STDOUR / ERR Vertical AUC lines Autoscale Y axis This test is based on Merlin s PedStats program. This program has strong limitations. First, any pedigree structure is disregarded by the PedStats software. If you analyze just large pedigrees the calculated significance level will be of no use to you because you used dependent individuals. Second, only a χ 2 significance test will be performed but no exact HWE test (Fisher statistics). Therefore, the test overestimates the significance in particular when dealing with small samples. On of our future project will be the substitution of this routine by completely newly programmed HWE modules. However, if you apply the test anyways you will be able to ignore the results or exclude markers that are apparently in HWD from subsequent linkage analyses at different significance levels (P < 0.05, P < 0.01, P < 0.001). In addition, HWE test results will be plotted on the final graphs as little tick marks in different colors (according to the significance) at the marker positions. This option activates sound outputs. In addition to the result files easylinkage generates after each run, this option shows the results in table form in the final results window. If you analyze large data sets the generation of this output which has indeed no particular use than just showing a table at the end of a run can significantly increase the required time. Therefore, it is usually recommended to leave this option switched off. As the sounds and tables options this option adds no specific functionality. It will just redirect any text output to a graphical window. The win32 console output is still there, however, you will not see any progress or error indicator in the console window. As default for multipoint linkage analyses easylinkage generates plots where the analysis points (mostly referred to as steps ) between two neighboring markers will be reflected as vertical lines. This is sometimes useful. However, if you analyze many markers on a chromosomes with small intermarker distances you will not be able to differentiate between individual analysis points anymore. As default easylinkage will generate plots where only LODs, NPLs, and other parameters >0 will be visible on the final plot. Some users might find it useful the enable the display of negative results as well. In this case the option must 26

27 be switched on. The Y axis will be scaled according to the results of the run for values > and < 0. 27

28 Options of linkage programs 1. FastLink The selection of Dominant or Recessive changes the penetrances in the lower part of the window automatically to standard values. In addition, penetrances can be edited individually. If you have loops in your pedigree you must check the loop option otherwise the program will terminate. Upon activation loops will be broken automatically ( unknown l ). A so called loop definition file is not required anymore. In general, we recommend using SuperLink that does that stuff without user actions. If you want to analyze for >1 liability class you must provide a liab file, activate the >1 liability class option and select the liability class file of your choice. The selection of the allele frequency algorithm is self-explaining. Additional descriptions can be found in the General notes chapter. In this example screen the active option Only TOTALS plot calculates only LOD scores over all families encoded in the PEDFILE. Appropriately, just one total plot of results will be provided by the program. In the case, the user wants to analyze families separately and together the option Plots for all families must be checked. The number of families is not limited. However this process can become very time-consuming depending on the number and sizes of the families. 28

29 2. SuperLink In general, SuperLink options are the same as for FastLink. SuperLink runs much faster than FastLink. The FastLink loop bug (if there is one pedigree with a loop only the TOTALS LOD will be calculated but not those of the individual families) does not occur in SuperLink. In this example, a SNP project was selected (dominant, reference allele frequencies from a Caucasian population provided by Affymetrix Inc.). If it comes to two-point analyses, we do always recommend SuperLink. It can also handle larger pedigrees than FastLink. 29

30 3. SPLink Only if you are familiar with that change the options according to your needs. 30

31 4. GeneHunter and GeneHunter Plus with/without ASM GeneHunter, GeneHunter Plus, GeneHunter Imprinting do all basic analyses providing LODs, NPLs, p values etc. In addition Genehunter Plus offers additional NPL analyses under a LIN or EXP model using the ASM program (Kong & Cox, Nature Genetics 1999). If you want to use GeneHunter Imprinting you must provide four penetrances for autosomes and 4/3 penetrances for females/males on the X chromosome. The as blue noted penetrance is only required for imprinting analyses. The other three penetrances must be provided if analyzing without the assumption of imprinting. 31

32 As mentioned before several analyses will not be available by default (GeneHunter Imprinting/TwoLocus). Any option that refers to these programs will only be visible if those programs are installed and the entries in the INI file are correct. The Haplotyping option increases the runtime of analyses significantly. Haplotypes will be delivered as ASCII files and as PostScript plots of the pedigree including markers and marker positions. This is an extension of the original GeneHunter PostScript plot. Plots for NPL, LOD, P Value, InfoContent are generated by easylinkage. GeneHunter Plus generates plots for Zlr, dhat score, MLOD for ASM lin and/or ASM exp in addition. GeneHunter zeroes the position of the first marker of a chromosome. This GeneHunter zero error will be corrected by easylinkage. For large scale SNP data it is recommended to analyze blocks of markers (here set to 100). The Compute sharing statistics should be deactivated whenever possible since that analysis part requires the most processing time. GeneHunter TwoLocus: GeneHunter TwoLocus performs parametric and nonparametric linkage analyses considering two loci at once. In order to perform parametric analyses you must provide a 4-by-4 pene- 32

33 trance matrix. If you model a nonimprinting situation penetrances m/+ and +/m must be identical (12 penetrance values in total). For further information please refer to the GeneHunter TwoLocus manual that comes with the software. For nonparametric analyses the values of the 4x4 penetrance matrix are irrelevant. 33

34 5. Allegro Allegro is an extension of GeneHunter. It does also provide allele sharing modeling as introduced by Cox and Kong. Allegro runs much faster than GeneHunter and analyzes slightly larger pedigrees. Allegro is also not limited in terms of the number of markers. However, using large quantities of SNPs can lead to a wrong haplotype assignment. In the worst case scenario a significant LOD score can just disappear. We recommend our guidelines as outlined in the General notes section. If you want to perform conditional nonparametric analyses you must provide weight files as outlined in chapter IV.4. 34

35 Another specialty of Allegro is the simulation of multipoint linkage data plus subsequent analyses of your defined model using the simulated data. Here some brief explanations of the simulation options: Marker number You must provide a number>0. We have experienced problems when using more than markers. However, it might still work in your setup. Marker DIST Here you provide an intermarker distance in cm (>0.001 cm). easylinkage will convert this distance into a recombination fraction. The option UNIT centimorgan does not work in this release of Allegro. Replicates Disease locus at cm Genotyping rate Heterogeneity We recommend using at least 10 replicates. That means that the pedigree information file you provide will be simulated 10 times. Finally, you will get 10 plots of 10 different simulated datasets with the same pedigree structure. If this option is set to 0 the disease locus will be different for each of the simulated replicates. In such a situation each simulated replicate would show maximum LOD peaks at different loci. Assuming you have several families with an identical phenotype and you want also a total LOD score over all families than you must provide a disease locus >0 cm. Then each replicate will peak at this particular locus and the TOTALS calculation will demonstrate summed LOD scores. In real life you will always have blanks in your data set in this regard means no drop-outs means 96% valid genotypes and 4% blanks. Heterogeneity can be modeled here means no heterogeneity. MIN / MAX numbers of alleles This means that every marker of your simulated data will have at least MIN and MAX alleles. In case of SNP data you should set this option to 2/2. In terms of microsatellites MIN could be >2 and MAX<10. If you activate the random generator than every marker will have randomized allele numbers >MIN and <MAX. 35

36 6. Merlin In principle all options are self-explaing. From version on Merlin can analyze parametric traits as well as nonparametric LIN and EXP models. Further, easylinkage makes use of Merlin s QTL capabilities. A QTL file containing the IDs and quantitative parameter must be provided. Please refer to section IV. Input files. 36

37 7. SLink The options are self-explaining. If you activate the Plot pedigree information file option then the pedigree structure file will be plotted on the final output of the ELOD charts. 37

38 8. SimWalk Most SimWalk options are self-explaining as well. Only parametric and nonparametric analyses will produce plots, all other analyses will only generate text files as output. If haplotyping is activated haplotyping files will be generated as well as map files and further input for HaploPainter. 38

39 VII. Output 1. Effects of options that can be set in the main screen of easylinkage 1.1 Mendelian test If you have downloaded PedCheck and activated it all genotyping files will be screened for Mendelian errors. In case, the files contained errors the following screen will appear: Print (LPT1): View Mendelian errors: Prints the report to the parallel printer port LPT1. You can look at important details of the errors: Remove Mendelian errors and continue: Self-explaining. In order to get a clean SNP file for additional runs activate the appropriate option. However, be careful: If you activated just one chromosome in the main screen only this chromosome 39

40 will be tested for Mendelian errors. It follows that the newly generated SNP file will only be cleaned for Mendelian errors on the activated chromosome. Therefore, you should activate all chromosomes if you want to inactivate the errors-containing SNPs for the entire SNP file. If you analyze microsatellite files removing of Mendelian errors means a complete disregard of problematic markers. 1.2 Non-Mendelian test This test checks for example whether a marker generates double recombinants within a haplotype. This particular marker may not show any Mendelian problem if analyzed as a single marker. The Non-Mendelian test is Merlin based and depends on the allele frequency algorithm and on the haplotype assignment by Merlin. In this way detected unlikely genotypes can be zeroed by clicking the appropriate button. If the user assumes wrong haplotype assignments done by Merlin the user can disregard those Merlin recommendations. 1.3 HWE (Hardy-Weinberg-Equilibrium) test As mentioned earlier in this manual this test is Merlin based and has its own pitfalls. First, Merlin disregards any pedigree structure. That means that dependent people (for instance members of one family) will be taken as independent. Second, the test is only a Pearsongoodness-of-fit χ 2 test. This test is not suited for smaller samples. Unfortunately, Merlin does not provide an exact Fisher test. Therefore, test results must be handled with care! If the user decides to disregard HWE errors test results will be displayed on the final plots as tick marks under the marker coverage lines. The color of those tick marks will correspond with the significance level of the HWE test. A legend will be plotted on the final output. 40

41 Final plot without displaying HWE test results: Final plot with displaying HWE test results: 41

42 1.4 Redirect STDOUT/ERR This option deactivates the console output and redirects it to an internal window. 1.5 Vertical AUC lines Activating this option results in displaying all analysis points between markers. 42

43 Option activated: 1.6 Autoscale Y axis If activated the y axis will be generated according to your results. All values <0 will be shown this way. Option activated: 43

44 Option deactivated (only values>0 will be shown): 44

45 2. Results table All calculated scores will be displayed as tables and as plots for every single parameter. Here, we present just the general output that differs between the linkage programs. Tables will be saved in plain text format, plots as PostScript files. The family ID column reflects the family IDs. In this regard TOTALS means that the LODs of all tested families will be added or averaged depending on the used programs. Top LODs table 45

46 This table reflects the LOD scores in descending numerical order. Note that this table is only shown in single-point analyses such as FastLink and SuperLink. 3. Intermarker distance map This table is shown in all analyses but SLink. It is useful if an investigator needs to make his genotyping map denser. It will be immediately visible where bigger gaps can be found. 4. Plots for linkage analyses 46

47 This is the general output window. Several plots and tables can be selected. All tables and plots are saved in plain text and PostScript files, respectively. The vertical red lines on the x axis represent the typed markers. Only when a marker resulted in a LOD > 0 the LOD will be shown as a vertical bar. All other LOD scores will be zeroed to generate a better visualization of the results. Project: The name of the working directory is used. Every directory level above the working directory will be removed automatically by the program. All other details are self-explaining and reflect the user entries. FastLink plots 1. Single chromosomes 47

48 2. Genome scan SuperLink plots 1. Single chromosomes 48

49 2. Genome scans SPLink plots 1. Single chromosomes 49

50 2. Genome scan 50

51 GeneHunter plots Besides the usual GeneHunter plots the following plots will be generated: I. Computations on a single chromosome 1. NPL plot 2. LOD plot 51

52 3. P value plot 4. InfoContent plot In GeneHunter Plus scores between markers can be higher than scores at the marker position! In this case the table in the upper part of the output reflects marker intervals rather than individual markers. For GeneHunter Plus the following additional plots will be generated: 52

53 5. NPL plots for ASM lin and ASM exp Both plots are identical with the GeneHunter NPL plot. 53

54 6. The newly weighted NPL score (Zlr plot) for ASM lin and ASM exp 54

55 7. The maximized LOD score (not identical with NPL or two-point LOD score!) for ASM lin and ASM exp 55

56 8. dhat plot for ASM lin and ASM exp 56

57 II. Genome scan 1. NPL plot 2. LOD plot 57

58 3. P value plot 4. InfoContent plot 58

59 5. NPL plots for ASM lin and ASM exp (identical with GENEHUNTER NPL plot) 59

60 6. The newly-weighted NPL score (Zlr plot) for ASM lin and ASM exp 60

61 7. The maximized LOD score (not identical with NPL!) for ASM lin and ASM exp 61

62 8. dhat plot for ASM lin and ASM exp 62

63 9. Pedigree plot and haplotypes An active Haplotyping on option results in the generation of an individual TOTALS plot and single haplotype plots for all used pedigrees. The original GENEHUNTER plot is modified in the following way: 1. Deletion of the list of haplotypes in the upper left part of the plot. 2. In every generation the markers with their positions will be displayed. 3. Additionally, the used genetic map is shown. Here is an example: 63

64 10. GeneHunter TwoLocus plots Those plots will be provided as postscript files that will be generated by GnuPlot (provided with easylinkage). 3D LOD plot 3D NPL plot 64

65 3D information content score plot 65

66 Allegro plots I. Single-point analyses I.1 Parametric analysis LOD score (in this example no single LOD>0) hlod score 66

67 Alpha score I.2 Nonparametric LIN analysis LOD score 67

68 NPL score Zlr score 68

69 dhat score I.3 Nonparametric EXP analysis LOD score 69

70 NPL score Zlr score 70

71 Exact P value for NPL Exact P value for LOD 71

72 Info content score dhat score 72

73 II. Multi-point analyses II.1 Parametric analysis LOD score (in this example no single LOD>0) 73

74 hlod score Alpha score 74

75 II.2 Nonparametric LIN analysis LOD score NPL score 75

76 Zlr score dhat score 76

77 II.3 Nonparametric EXP analysis LOD score NPL score 77

78 Zlr score Exact P value for NPL 78

79 Exact P value for LOD Info content score 79

80 dhat score Plots are generated as TOTALS plot or plots for individual families. 80

81 5. Plots for simulation studies Two-point simulation analyses with FastSlink Plots/output This version of easylinkage will break any loop automatically which is an extension of the original program. The final plot of a simulation will show the expected LOD scores (ELOD) for different intermarker distances. Note, that the pedigree structure file will be plotted as well. 81

82 Two-/Multipoint simulation analyses with Allegro I. General notes for Allegro based simulation studies The introduction of multipoint simulation analyses plus subsequent linkage analyses is a novelty of easylinkage. By using Allegro it is possible to perform two-point as well as multipoint linkage analyses with parametric/non-parametric inheritance models. In particular for simulating a pedigree structure for SNP projects the program turned out to be very useful. In our understanding we model a 10k chip project as follows: to 800 SNPs. You can even model or more SNPs. However, an average 10k-SNP scan would usually not contain more than 800 markers per chromosome. Using more markers extends the computation time but does not yield more information. 2. Intermarker distance = 0.33 cm (assuming SNPs for a total genome length of cm) 3. Genotyping rate = We recommend using at least 10 replicates to get an impression of the variation of ELODs. 5. If you have several pedigrees in your pedigree information file where you observed identical phenotypes and where you suppose the same genetic defect we recommend to use a disease locus >0.00 cm. If the disease locus stays 0.00 cm easylinkage assumes that each pedigree has a different genetic background. On the following pages we present an example anlyses. The following parameters where in effect: 1. Marker number = Marker distance = 0.33 cm 3. Replicates = Disease locus at 80 cm 5. Genotyping rate = 1.00 (no blanks in your data) 6. Heterogeneity = Min/Max = 2/2 Plots for the following parameters can be generated: 1. parametric LOD 2. nonparametric LOD 3. LOD P values 4. NPL 5. NPL p values 6. Zlr 7. Information content scores 8. Alpha 9. dhat 10. HLOD Here, we present only a part of the thinkable possibilities. 82

83 II. II.1 Two-point simulation Parametric models => 3 replicates of 1 pedigree (note the different results): 83

84 => TOTALS for 10 replicates with a fixed disease locus: 84

85 II.2 Non-parametric models II.2.1 LIN => 3 replicates for 1 pedigree: 85

86 => TOTALS for 10 replicates with a fixed disease locus at 80 cm: 86

87 II.2.2 EXP => 3 replicates for 1 pedigree: 87

88 => TOTALS for 10 replicates with a fixed disease locus at 80 cm: 88

89 III. Multipoint simulation III.1 Parametric models => 3 replicates for 1 pedigree: 89

90 => TOTALS for 10 replicates of 1 pedigree with a fixed disease locus at 80 cm: 90

91 III.2 Non-parametric models III.2.1 LIN => 3 replicates of 1 pedigree: 91

92 => TOTALS for 10 replicates of 1 pedigree with a fixed disease locus at 80 cm: 92

93 III.2.2. EXP => 3 replicates of 1 pedigree: 93

94 => 10 replicates of 1 pedigree with a fixed disease locus at 80 cm: 94

95 IV. Selection of plots for other parameters NPL (linear model, two-point analysis) Information content score (linear model, two-point analysis) 95

96 NPL (exponential model, two-point analysis) Information content score (exponential model, two-point analysis) 96

97 NPL (linear model, multipoint analysis) Information content score (linear model, multipoint analysis) 97

98 NPL (exponential model, multipoint analysis) Information content score (exponential model, multipoint analysis) 98

99 Notes to the program HaploPainter easylinkage generates input files for the program HaploPainter if running any multipoint analysis with a switched on Haplotyping option. HaploPainter needs three files (here we show an example for chromosome 1): 1. Pre- or post-makeped pedigree information file N N N N N Haplotyping file B F Y A B S S S S S S S S S S S S S S S S S S S S S S S S S T S S S S S S S S M S S S S S A F D D D D D D D D D D D D D D D D D D D D D D D D D G D D D D D D D D A D D D D D N N N N N N N N N N Marker map file #Chr Genpos Marker Physpos Nr D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S D1S

100 D1S GATA124F D1S D1S D1S D1S D1S D1S D1S D1S AFMB361YB D1S D1S D1S D1S D1S2682 A plot like the following will be generated: The left panel shows the typed marker with the appropriate marker positions in cm. The program can be obtained from: Author: Holger Thiele 100

101 VIII. Special case: SNP project plots for pedigrees containing loops These section is to demonstrate the power of multipoint analyses versus single-(two-)point analyses. A pedigree with 2 loops was analyzed with SuperLink, FastLink, and Allegro (SPT and MPT PAR). 581 informative SNPs were tested on chromosome 5. Here are the results: SuperLink: SuperLink runs much faster than FastLink. Running time: 6 min 32 s FastLink: Even two-point linkage programs might pickup LOD scores in case of reduced SNP informativity if the pedigree was large enough. Running time: 10 min 9 s 101

102 Running time: 3 min 44 s Allegro single-point analysis (parametric): Allegro multi-point analysis (parametric): LOD scores sharpened up. Background noise disappeared. Running time: 3 min 44 s As mentioned earlier, haplotypes can be assigned wrongly when using large scale SNP data. Therefore, we implemented the option of analyzing chromosomes with defined sets of markers. The user should run the entire analysis at least twice with different set sizes. When switching on this option the output will contain additional blue vertical lines along the marker coverage panel indicating the end of the previous and the beginning of the next marker set. It will look as follows: 102

103 Genome-wide scan Single-point Analysis Genome-wide scan Multi-point Analysis 103