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USER MANUAL VERSION 3.2

1. P aL aM Figure 4 Manhattan plot setting for pixel sizes and chromosome colors upper and Manhattan plot with threshold P value line lower Unknown Chromosome 32 Run Save Graph s Current Graph l Current Trait All Graphs Manhattan Plot TRAITOO Results SNP All Traits TRAITOO ManhattanPlot I1 p1 P1 90 Results SNP All Traits TRAITOO Q QPlot Results SNP All Traits TRAITOO 1 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 2 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 3 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 4 I1 i1 p1 P1 90 Results SNP All Traits TRAIT00 5 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 6 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 7 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 8 11 il p1 P1 g0 Results SNP All Traits TRAITOO 9 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 10 11 i1 p1 P1 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome Cut off P value 4e 7 Threshold P value Line 4e 10 Shade Points below Threshold Line Line needs to be off W Unknown Chromosome 32 Save Graph s Chromosome Current Graph Current Trait All Graphs oo LENT 9 o ee s t ove a nin atin Bas d qu ee doum qo
2. ot t Original Value Sr No x a fate t Te E usn te z T Y Rw yr MT p atte 1d vir Je t yr M o on Akt foes y t n _ arx lever e 0 E39 F9 C3 Sl T Unknown Chromosome Save Graph s Chromosome Figure 3 Manhattan plot displaying the log10 1 p values of genetic effects upper and Manhattan plot displaying original values lower 21 The upper right window of SNPEVG2 in Figure 3 is the Graph list showing a list of graphs produced by the Run button Manhattan plot of each genetic effect and original value 1s listed and is followed by the Q Q plots and the chromosome graphs The user can switch between graphs in the graph list by using the up or down arrow key For example using the down arrow key at the first line of the list the program displays the Manhattan plot of the next genetic effect EFFECTO1 Similarly the up arrow key displays the previous Manhattan plot for EFFECTOO Pixel Size Fixed size Dynamic size Min Chromosome Color Template 1 Template 2 Template 3 L L Template 4 E Template 5 m in BE E Manhattan Plot EFFECTO5 Results MixedValues All ManhattanPlot EFFECTOO I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO1 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO02 I1 p10 p20 P Results MixedValues All ManhattanPlo
3. Figure 13 Effect setting dialog for copying one effect s color to another upper and chromosome graph with the blue color for Y1 axis and the purple color for Y2 axis lower If Original value is checked the Y axis prints the original P value rather than log 1 P and the user may change Original value to a user specified title 30 4 4 Save Graphs to png Files The program saves a graph to an image in the same folder of the input file by the graph s actual dimension A selected graph can be saved to a png file by clicking Current Graph and all the graphs can be saved to png files by clicking All Graphs 31 Part III SNPEV Gconvert Input format conversion of output file of any GWAS software to input file for SNPEVGI1 and SNPEVG2 Contents 1 2 3 4 Oe CION m M LU o MEN SET M LO NE E FILE FORMA Ro er RUNNING SNPEVGCONVERT Derisinin niaire einean SEEN URS EP ri aa EE ERE EAAUE NS ERR AP EREE caasisesstsoeanieiss 22 1 INTRODUCTION SNPEVGconvert is a tool for input format conversion from output file of any GWAS software to the input file format for SNPEVG The program provides the user the flexible control of file format conversion for SNPEVGI and SNPEVG2 for the necessary data and user selected P values 2 INSTALLATION 2 1 Windows x86 x64 The installation of SNPEVGconvert only requires to create a folder
4. Figure 7 Manhattan plot with user s color selection upper and Manhattan plot with cut off P value and shading of data points below threshold P value line lower In the Manhattan plot each chromosome width is the true width of the chromosome defined by the starting and ending SNP marker positions The Manhattan plot has the options to change the setting of pixel sizes and colors by clicking the icon beside its checkbox Figure 4 and 7 The setting of pixel sizes has two options The fixed pixel size allows the user to change the size of pixels between 1 and 9 The dynamic pixel size allows the pixels to be varied according to the data points significance within the user defined range The color of each chromosome in Manhattan plot is assigned according to the selected color template The template colors are 10 extended by rotation and can be changed by double clicking the region of a color to select a new color or moving a color by dragging and dropping the color region in the same template Manhattan plot also has options to mark the threshold P value line for declaring significance Figure 4 to show data points above the cut off P value Figure 5 or to shade data points below threshold P value line Figure 6 and 7 4 3 Q Q Plot for all SNP Effects In the GUI of the upper figure in Figure 2 check Q Q Plot and the program will produce a Q Q plot Figure 8 shows the Q Q plot using all P values Warning If a cut o
5. Chromosome 3 Mb Figure 10 Chromosome graph with threshold P value line upper cut off P value lower 13 4 5 Save Graphs to png Files The program saves a graph to an image in the same folder of the input file by the graph s actual dimension A selected graph can be saved to a png file by clicking Current Graph all the graphs of the currently selected trait can be saved to png files by clicking Current Trait and all the graphs of all traits can be saved to png files by clicking All Graphs 14 Part II SSNPEVG2 Multiple traits values graph Any method of significance testing Any values of variables Contents 1 INTRODUCTION occscscsscsscsscssssssssssssesssssssesssssssessssssssssssssssssessssessessssessesssssssessssesscsussessesessessesssssessessesessessessees 16 2 TING A OWN 2 ornnes esa cece ceca ators crt sees AETA seta sce DEN CE 17 3 INPUT FILE FORMATS scot cs vas cctccesc aces vncocspavcascapvecccensvatetesscesecazsectssasseccstessvstsdnieatsnpcsnsanasactnateen 17 A RUNNING NV O eai entera pre cesta E E Ie TT nln S 18 1 INTRODUCTION SNPEVG2 is a graphical tool for the viewing and graphing of multiple traits on the same graph with the large quantities of genome wide test results The program currently uses the same input file SNPE of SNPEVGI for drawing figures Each chromosome figure can display multiple genetic effects using P values from any methods of significance t
6. Current Graph Current Trait or All Graphs e Each Manhattan plot has the following options and features Each chromosome width 1s the true width of the chromosome defined by the starting and ending SNP marker positions Adjustable fixed pixel size and dynamic pixel size Color templates with the support of custom colors Horizontal line marking the threshold P value for declaring significance Display of P values above the specified cut off P value Shading P values below the threshold P value line e Each chromosomal figure of P values has the following options Horizontal line marking the threshold P value for declaring significance Display of P values above the specified cut off P value Shading P values below the threshold P value Optional use of lines to connect data points User specified unknown chromosome that will plot P values in the sequential order of SNP markers rather than chromosome positions e Q Q plot for each trait displaying all data points 2 INSTALLATION 2 1 Windows x 6 x64 The installation of SNPEVGI only requires to create a folder to place all files in SNPEVG zip in that folder and then change SNPEVGI exe file to SNPEVGlI exe file The Windows x64 64 bit version 1s available by request 2 2 Mac OS X 10 6 or newer 64 bit The installation of SNPEVGI only requires to unzip SNPEVGI Mac zip file to obtain SNPEVGI app file 3 INPUT FILE FORMATS One input file with the ex
7. log10 1 p amp Original Value m Pe errectoo E 2 eve I e IM owe we ece Fal aee v r1 E errecros ipd ees WIE 0 e ex 0 Wim cs IBI emi MMB eee MEE ce il eco Cc Co tvi e NE Ir oen FI IER FI cee fa I ec e vr cue IL TETTE JE IE EL v ec Ult M Figure 2 Upload of an input file upper value and axis setting to display lower left and color selection dialog for values lower right 20 Manhattan Plot EFFECTO5 Results MixedValues All ManhattanPlot EFFECTOO I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO1 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO02 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO3 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO04 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOS I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO6 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO7 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOS I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO9 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT10 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT11 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT12 I1 p10 p20 P n H Input File IPEVG Results MixedValues Allsnpe Browse logi0 1 p Yi 16 Y2 0 15 Advanced Options Manhattan Plot gt v Q Q
8. oe Deere X aV insti tud A v t Manhattan Plot EFFECT05 ee v of e Sy ve er A e 99 9 tet t 4 ad ad wet ZTAANtee Seer v A n EP rer Chromosome Results MixedValues All ManhattanPlot EFFECTOO I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTOZ1 I1 p11 p20 P E Results MixedValues All ManhattanPlot EFFECTO2 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO3 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO04 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTOS I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO6 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO7 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO8 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO9 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT10 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT11 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT12 l1 p11 p20 P r Input File IPEVG Results MixedValues All snpe Browse Y1 146 Y2 0 15 Advanced Options Manhattan Plot 3 V Q Q Plot Line SNP Position Chromosome S9 3 3 K Cut off Value Threshold Value Line 1 0E 10 4 0E 23 Significance log10 1 p Manhattan Plot EFFECT05 Fase EL eee MAC tee ILE v E f pre gm Mure VETUS S mem ymo amp m N m c
9. Results MixedValues All ManhattanPlot EFFECT12 I1 p10 p20 P 7 r Shi eee e he codi acr 1o y em s 23 Input File IPEVG Results_MixedValues_All snpe Browse Effect s Tota 31 Y1 16 Y2 oy 15 Advanced Options 7 Manhattan Plot 3 V Q Q Plot Line SNP Position Chromosome Cut off Value V Threshold Value Line 4 0E 23 Shade Points below Threshold Line Line needs to be off W Unknown Chromosome 32 Save Graph s Figure 6 Manhattan plot with shading of data points below threshold value line Pixel Size Basic colors Fixed size 2 LE mE E e Ei z E HEHNENMENLZN Dynamicsize Min 1 E ii imr C I C iF Ei ENENMNENUN Chromosome Color Template 1 O Ttm BED Tmas MORN Custom colors ec I NEL NN DN LILIL IL IE IL IL IL I Tmas FR HR LIL IL JE JE IE JL ILI Manhattan Plot EFFECTO5 Results MixedValues All ManhattanPlot EFFECTOO I1 p11 p20 P H n z Results MixedValues All ManhattanPlot EFFECTO2 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO3 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO04 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTOS5 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO6 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO7 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECTO8 I1 p11 p20 P Results MixedValues All Man
10. W Manhattan Plot 7 Q Q Plot V Line J SNP Position e LIP E iy we o utn gt SANA ASOR I d Vay onde og x as ko i ee nt D h s d NGA eae wets Me Lt mam Am m nn LL 7 Amm e Mihri ad A D 9 e Pte ad drm getse vied ae ni 2 px 4 Y H 7 b e A Chromosome Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off 4 Unknown Chromosome 32 Run Save Graph s Chromosome Current Graph Current Trait All Graphs Figure 3 Manhattan plot The left window of Figure 3 is the Graph window for viewing graphs The Manhattan plot is always the first graph in the list of each trait s graphs and the first graph is the default display of all graphs when the user switches the trait The upper right window of Figure 3 is the Graph list showing a list of graphs produced by the Run button Line 1 is the Manhattan plot line 2 1s the Q Q plot and the rest are chromosome graphs of the trait The user can switch between traits using the left or right arrow key For example using the right arrow key at the first line of the list the program displays the Manhattan plot of the next trait TRAITOI Similarly the left arrow key displays the previous Manhattan plot for TRAITOO The up or
11. column 3 is the marker position and the first row is the header of the file Marker name chromosome number and marker position start in the second row 8 RUNNING SNPEVG3 To run SNPEVG3 open the folder that holds the SNPEVG3 files and required input files Double click SNPEVG3 exe For Mac users double click SNPEVG3 app will open a graphical user interface GUI of SNPEVG3 as shown below Figures in the manual are produced in Windows 7 2 SNP Effect View and Graph Input File Browse Browse SNP Position SNP Effects V Genotypic Significance 7 Dominance Significance V Additive Significance Number of Observations Advanced Options 7 Manhattan Plot gt 7 Q Q Plot V Line E Chromosome E Trait C Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome C Sort SNP Position needs to be off 9 Genotypic Significance Dominance Significance Additive Significance Number of Observations l Run Save Graph s l Current Graph Current Trait All Graphs Figure 1 Graphical User Interface of SNPEVG3 37 Genotypic Significance Manhattan Plot Trait1 n rm x single locus fig Traitl ManhattanPlot l0 pO0 P0 g0 mg single locus fig Traitl ManhattanPlot l0 p0 P0 g0 ma single locus fig Traitl ManhattanPlot I0 pO P0 g0 md single locus fig
12. genotypic additive and dominance effects and the sample size for each SNP AII options for chromosome figures of SNPEVGI are available for SNPEVG3 Examples are shown in Figures 6 4 3 Sort SNP Effects with Position Option off SNP effects can be sorted according to the significance of one effect given that the position option is turned off Figure 7 4 4 Save Graphs to png files The program saves a graph to an image in the same folder of the input file by the graph s actual dimension A selected graph can be saved to a png file by clicking Current Graph all the graphs of the currently selected trait can be saved to png files by clicking Current Trait and all the graphs of all traits can be saved to png files by clicking All Graphs 43 AUTHOR CONTRIBUTIONS Shengwen Wang is the author of the SNPEVG3 program Daniel Dvorkin is the author of the EPISNPPLOT program that is partially used in SNPEVG3 Yang Da is the project leader and the lead writer of this user manual RELEASE HISTORY SNPEVG version 3 2 Released by S Wang D Dvorkin and Y Da November 2012 SNPEVG version 3 1 Released by S Wang D Dvorkin and Y Da June 2012 SNPEVG version 2 1 Released by S Wang D Dvorkin and Y Da June 2011 SNPEVG version 1 1 Released by S Wang D Dvorkin and Y Da February 2011 REFERENCES Ma L Runesha HB Dvorkin D Garbe JR Da Y Parallel and serial computing tools for testing sin
13. GVALUE03 GVALUEO4 GVALUEOS O GVALUE06 GVALUEO7 t GVALUE08 Z GVALUEO9 GVALUE10 OGVALUE11 GVALUE12 x GVALUE13 Y GVALUE14 anyer PUIBLIO Results MixedValues All 1 I1 0 p11 p21 P11 P21 Results MixedValues All 2 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 3 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 4 I1 10 p11 p21 P11 P21 90 Results MixedValues All 5 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 6 I1 i0 p11 p21 P11 P21 90 Results MixedValues AIl 7 I1 10 p11 p21 P11 P21 90 Results MixedValues All 8 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 9 I1 10 p11 p21 P11 P21 90 Results MixedValues All 10 I1 10 p11 p21 P11 P21 90 Results MixedValues All 11 I1 10 p11 p21 P11 P21 90 Results MixedValues All 12 I1 10 p11 p21 P11 P21 90 Results MixedValues All 13 I1 10 p11 p21 P11 P21 90 4 Input File IPEVG Results MixedValues Allsnpe Effect s Tota 31 Y1 y 16 Y2 o 15 Advanced Options E Manhattan Plot E Q Q Plot Line SNP Position Chromosome Cut off Value 1 0E 11 15 Threshold Value Line 1 0E 26 37 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Rn 0 Save Graph s Chromosome 1 Mb X GVALUEOO V GVALUEO1 A GVALUEO2 GVALUE03 GVALUE04 GVALUEOS O GVALUE06 amp GVALUE08 T GVALUEO9 GVALUE10 OGVALUE11 GVALUE12 x GVALUE13 Y GVALUE14 Results MixedValues All L IL iQ p11 p21 P11 P21 90 Results MixedValues Al
14. Line needs to be off Unknown Chromosome 32 Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Lo Rn E Save Graph s Chromosome 01 Mb O Genotypic Significance O Additive Significance Dominance Significance x Number of Observations single locus fig Traitl ManhattanPlot I1 p1 P1 g0 mn single locus fig Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md single locus fig Trait1 01 I1 i0 egadn p1 P1 90 s0 single locus fig Trait1 02 I1 i0 egadn p1 P1 90 s0 Input File C SNPEVG single locus fig out SNP Position C SNPEVG snp_position pos SNP Effects Genotypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options Manhattan Plot gt 7 Q Q Plot Line Chromosome E Trait Cut off P value 0 0000004 Threshold P value Line 00000004 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Lo Rn 0 jJ Save Graph s K Chromosome 01 Mb Figure 6 Example of chromosome graph with cut off P value and threshold P value using lines upper or symbols lower 42 Additive Significance er single locus f
15. M7 0 184F 01 0 180E 01 0 803E 00 290 trait 19 M7 0 103E 01 0 BSE OL 0 220E 00 290 trat 19 M8 0 398E 00 0 601E 00 0 347E 00 295 trait2 19 M8 0 188E 00 0 413E 00 0 121E 00 295 trartl 19 M9 0 763E 00 0 930E 00 0 663E 00 295 trait2 19 M9 0 105E 01 0 122E 00 0 145E 01 295 traiti 19 M10 0 170E 01 0 174E 01 0 967E 00 292 trait2 19 M10 0 782E 00 0 842E 00 0 697E 00 292 traitl 19 M11 0 692E 00 0 345E 00 0 940E 00 295 trait2 19 M11 0 473E 00 0 113E 00 0 841E 00 295 traiti 19 M12 0 396E 00 0 521E 00 0 434E 00 295 trait2 19 M12 0 119E 00 0 115E 00 0 309E 00 295 In the above file column 1 1s the trait name column 2 is the chromosome number column 3 is the marker name column 4 is loglO 1 p values for marker genotypic effect column 5 is log10 1 p values for additive effect column 6 is logl10 1 p values for dominance effect and column 7 is the number of individuals with known genotypes for this marker With this input 36 file SNPEVG3 plots the logl0 1 p values in the sequential order of the markers on each chromosome A chromosome and position file is required if the user wants to plot the log10 1 p values according to the markers physical location defined either by base pair location or genetic distance in centi Morgans The file name s extension is pos The following is a short example of this position file SNP Chr Position M12 I 135098 M7 i 267940 In the above file column 1 is the marker name column 2 is the chromosome number
16. Plot Line SNP Position Chromosome Cut off Value Threshold Value Line Shade Points below Threshold Line Line needs to be off ee of Significance 4 lt Wr isan y Seg er A LT A we tet adi Eee LE Por i E M 3 3 E p I T Unknown Chromosome AMMEMNENNMONE SE 51 Save Graph s Manhattan Plot GVALUE11 Results MixedValues All ManhattanPlot GVALUEOS8 I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUEOO I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUE10 I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUEL1 I1 p10 p20 I Results_MixedValues_All ManhattanPlot GVALUE12 I1 p10 p20 f Results MixedValues All ManhattanPlot GVALUE13 I1 p10 p20 F L Results MixedValues All ManhattanPlot GVALUE14 I1 p10 p20 F Results MixedValues AII Q QPlot EFFECTOO Results_MixedValues_All Q QPlot EFFECTO1 Results_MixedValues_All Q QPlot EFFECTO2 Results MixedValues AII Q QPlot EFFECTO3 Results MixedValues AII Q QPlot EFFECTO4 Results MixedValues All Q QPlot EFFECTO5 m Input File IPEVG Results MixedValues All snpe Browse Effect s Total 31 Yt y 16 Y2 0 15 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome Cut off Value Threshold Value Line Shade Points below Threshold Line Line needs to be off ARIS Hs quum p Lu TT I e s e ZI as adu SONRISA Aaa 2d
17. Results MixedValues All 5 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 6 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 7 I1 10 p10 p20 P10 P20 90 Results MixedValues All 8 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 9 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 10 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 11 I1 10 p10 p20 P10 P20 90 Results MixedValues All 12 I1 10 p10 p20 P10 P20 90 Results MixedValues AlI 13 I1 10 p10 p20 P10 P 20 90 Input File IPEVG Results MixedValues All snpe Effect s Tota 31 Y4 16 Y2 o 15 Advanced Options E Manhattan Plot E Q Q Plot C Line SNP Position E Chromosome E Cut off Value E Threshold Value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 EFFECT06 CJEFFECT11 XGVALUEOO GVALUEOS F 4 x EFFECTO7 Vv GVALUEO1 GVALUEOO Results MixedValues All 1 I1 i0 p10 p20 P11 P21 g A GVALUEO2 GVALUE10 Results_MixedValues_All 2 11 10 p10 p20 P11 P21 g0 GVALUEO3 C GVALUE11 i X GVALUEO4 GVALUE12 Results MixedValues A1L3 1 0 p10 p20 P11 P21 g0 GVALUEOS lt GVALUE13 Results MixedValues All 4 I1 10 p10 p20 P11 P21 90 EFFECTO9 EFFECT10 aa Y GVALUE14 Results MixedValues All 5 I1 i0 p10 p20 P11 P21 90 Results MixedValues All 6 I1 10 p10 p20 P11 P21 90 Results MixedValues All 7 I1 10 p10 p20 P11 P21 90 Results MixedValues All 8 I1 10 p10 p20 P11
18. Results_SNP_All_Traits TRAITOO 5 IL i0 p1 P1 g1 Results_SNP_All_Traits TRAITO0 6 IL i0 p1 P1 g1 Results_SNP_All_Traits TRAITOO 7 1L i0 p1 P1 g1 Results_SNP_All_Traits TRAITO0 8 11 i0 p1 P1 g1 Results SNP_All_Traits TRAITOO 9 I1 i0 p1 P1 g1 Results_SNP_All_Traits TRAITOO 10 I1 i0 p1 P1 g1 Input File NPEVG Results SNP Al Traits snpe Traits Total 31 Display 31 Advanced Options V Manhattan Plot 3 V Q Q Plot Line SNP Position qe Ly Pil bd kaak L e c e s osto o O o ole ETO e Pe t Ws use sS s B uw wie art E a a 4 9 v t s ets 4 c wv TLt gt In t6 E w Az A a Se a xxr e Se P 3 d t d did PES x uel e r M M yw Aa Chromosome Cut off P value 4e 7 Threshold P value Line 4e 10 J Shade Points below Threshold Line Line needs to be off 4 Unknown Chromosome 32 Save Graph s Current Graph Current Trait Vea eae ter kl H ne Y amt ote acd ee qettal E ot uke P dod NO m Xt Ant dd I AE nt 3 ie t gt t wr oe LEO PLE p wh seg 2 ue ae A e VU ST Lc N s s qum kn Te t ro we ot ie eria Yate dv 2 Sad a Albee ow fet Do Duel ar w
19. SNPEVG A Graphical Tool For SNP Effect Viewing and Graphing Part I SNPEVGI page 3 Part II SNPEVG2 page 15 Part III SNPEVGconvert page 32 Part IV SNPEVG3 page 35 USER MANUAL VERSION 3 2 Shengwen Wang Daniel Dvorkin Yang Da Department of Animal Science University of Minnesota November 2012 Part I SNPEVGI Multiple traits one trait graph Any method of significance testing Contents l A3NIBODUELPION 22 enirn EUREN OIN HUE oO DE e Ee EU M E MEE 2 INSTALLATION cccsssssssssssssssssssessessssessscessosssssssssssessvssssessosssesssvssessssvssesessvssssessossssessossssossossssvssssvsssssssosseesees am Ds al 0 F021 0 21 0 0 emo ee ios E NE eE TRUE d RUNNING GN Gaia sere E see est A assesses VIE d EFESVEREEESS IMS 1 INTRODUCTION SNPEVGI is a graphical tool for SNP effect viewing and graphing for rapid digestion of large quantities of genome wide significance test results using P values from any methods of significance testing Assuming 30 traits and 30 chromosomes per trait one command of Run produces 960 graphs that will greatly facilitate the digestion of GWAS results The total number of graphs that can be generated by one Run is n c 2 where n is number of traits with 0 lt n lt 100 and c is the number of chromosomes Any of the graphs could be readily placed in a publication SNP effect viewing and graphing is accomplished through a user friendly graphical user interface GUI th
20. down arrow key allows switching between graphs in the graph list Pixel Size Q Fixed size e Dynamic size Min 1 Chromosome Color Template 1 Template 2 j Template 3 Template 4 7j Template 5 2 SNP Effect View and Grap TRAITOO Manhattan Plot TRAITOO Results SNP All Traits TRAITOO ManhattanPlot I1 pO P1 g0 Results SNP All Traits TRAIT00 Q QPlot Results SNP All Traits TRAITOO 1 I1 i1 pO P1 g0 Results SNP All Traits TRAITO0 2 I1 i1 p0 P1 g0 Results SNP All Traits TRAITOO 3 I1 i1 p0 P1 90 Results SNP All Traits TRAITOO 4 I1 i1 p0 P1 90 Results SNP All Traits TRAITOO 5 IL i1 p0 P1 g0 Results SNP All Traits TRAITOO 6 I1 i1 p0 P1 90 Results SNP All Traits TRAITOO 7 I1 i1 p0 P1 90 Results SNP All Traits TRAITO0 8 I1 i1 p0 P1 g0 Results SNP All Traits TRAITOO 9 I1 i1 p0 P1 90 Results SNP All Traits TRAIT00 10 11 i1 p0 P1 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options 7 Manhattan Plot 7 Q Q Plot V Line v SNP Position Chromosome eA o e Knut te oe gt a s E oom A om woane 4 v Cut off P value sat 4 Threshold P value Line 0 0000000004 ye P Shade Points below Threshold Line Line needs to be off Li AA At TR TB AN 09 PNE 009 08 O9 09 098 09 0 Chromosome
21. g0 Results_SNP_All_Traits TRAITOO 2 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 3 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 4 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 5 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 6 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 7 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 8 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 9 I1 i0 p0 P0 g0 Results_SNP_All_Traits TRAITOO 10 I1 i0 p0 P0 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Save Graph s Chromosome 1 Mb Figure 9 Example of SNP effects by chromosome with lines upper and with symbols lower 12 Results SNP All Traits TRAITOO ManhattanPlot l1 pO P1 90 Results SNP All Traits TRAITO0 Q QPlot Results SNP All Traits TRAITO0 1 11 i1 p0 P1 g0 Results SNP All Traits TRAITO0 2 11 i1 p0 P1 g0 Results SNP All Traits TRAITOO 3 IL i1 pO P1 g0 Results SNP All Traits TRAITO0 4 11 i1 p0 P1 g0 Results SNP All Traits TRAITOO 5 l1 i1 p0 P1 90 Results SNP All Traits TRAITOO 6 IL il pO P1 90 Results SNP All Traits TRAITOO 7 l1 i1 p0 P1 g0 Results SNP AII Traits TRAITOO 8 11 i1 p0 P1 g0 Results SNP All Traits TRAITO0 9 I1 i1 p0 P1 g0 Results SN
22. needs to be off v Unknown Chromosome 32 E Sort SNP Position needs to be off 8 Genotypic Significance Dominance Significance Additive Significance Number of Observations Save Graph s 5 34 8 01 10 68 13 35 16 02 18 69 21 36 24 03 26 70 Expected Significance log10 1 p Figure 2 Manhattan and Q Q plots by SNPEVG3 38 In the GUI of Figure 1 specify the input file s select the options and then click Run to produce the corresponding graphs for one or multiple traits Alternatively the user can drag the input file and drop to the corresponding input field without using browse button Pixel Size Fixed size Dynamic size Min 1 Chromosome Color Template 1 a Template 2 L Template 3 L K Le Template 4 L E Template 5 Ld E E Genotypic Significance Trait1 T Manhattan Plot Traiti single_locus_fig Traitl ManhattanPlot 1 p0 P1 g0 mg a single locus fig Traitl ManhattanPlot I1 p0 P1 g0 ma a single locus fig Traitl ManhattanPlot I1 p0 P1 g0 md single locus fig Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md Input File C SNPEVG single locus fig out V SNP Position C SNPEVG snp_position pos Browse SNP Effects V Genotypic Significance V Dominance Significance WV Additive Significance _ Number of Observations Advanced Opt
23. the chromosome defined by the starting and ending SNP marker positions The Manhattan plot has the options to change the setting of pixel sizes and colors by clicking the icon beside its checkbox Figure 4 and 7 The setting of pixel sizes has two options The fixed pixel size allows the user to change the size of pixels between and 9 The dynamic pixel size allows the pixels to be varied according to the data points significance within the user defined range The color of each chromosome in Manhattan plot is assigned according to the selected color template The template colors are extended by rotation and can be changed by double clicking the region of a color to select a new 24 color or moving a color by dragging and dropping the color region in the same template Manhattan plot also has options to mark the threshold value line for declaring significance Figure 4 to show data points above the cut off value Figure 5 or to shade data points below threshold value line Figure 6 and 7 Q Q Plot for all SNP Effects In the GUI of the upper figure in Figure 2 check Q Q Plot and the program will produce a list of Q Q plots for each genetic effect Figure 8 shows a Q Q plot using all P values Warning If a cut off value is specified the Q Q plot only displays values above the cut off value We do not recommend this option for Q Q plot RE SNP Effect View ond Graph 2S 1 n Q Q Plot EFFECTOO Results MixedValues All Manhat
24. 0 2 48E 03 1 11E 03 1 58E 03 2 74E 02 2 65E 01 SNPO2 1 845494 7 02E 04 9 07E 04 8 91E 05 2 56E 02 8 02E 02 SNPO3 1 883895 1 87E 02 1 71E 01 3 07E 03 1 07E 02 2 81E 03 SNPOA 1 929617 1 79E 03 1 57E 02 1 39E 05 9 03E 02 2 99E 04 SNPO5 1 950841 1 20E 03 7 07E 03 1 32E 05 8 46E 02 1 71E 03 For the given input file SNPEVGI plots the log10 1 p values according to the SNP positions on each chromosome for SNP markers with known chromosome positions or in sequential orders of the SNP markers for SNPs in the chromosome file specified as unknown by the user For each trait column SNPEVGI produces the following graphs e Manhattan plot with options e Q Q plot with one option e Graphs for one or all chromosomes with options 4 RUNNING SNPEVGI To run SNPEVGI open the folder that holds the SNPEVGI files and required input files Double click SNPEVGlI exe For Mac users double click SNPEVGI app will open the GUI of SNPEVGI as shown in Figure 1 below Figures in the manual are produced in Windows 7 Input File Browse Traits Total Display Advanced Options v Manhattan Plot 3 7 Q Q Plot V Line V SNP Position Chromosome C Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome l Run Save Graph s Current Graph Current Trait All Graphs Figure 1 Graphical User Interface GUI
25. Les Les SOS 53 E E EI CE CET 00000 PT TTT TTT TT Tf E CIGVALUE11 GVALUEL3 Results_MixedValues_All 1 11 i0 p11 p21 P11 P21 g0 Results MixedValues All 2 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 3 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 4 I1 10 p11 p21 P11 P21 90 Results MixedValues All 5 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 6 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 7 I1 10 p11 p21 P11 P21 90 Results MixedValues All 8 I1 10 p11 p21 P11 P21 90 Results MixedValues All 9 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 10 I1 10 p11 p21 P11 P21 90 Results MixedValues All 11 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 12 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 13 I1 10 p11 p21 P11 P21 90 4 Input File IPEVG Results MixedValues All snpe Effect s Total 31 Y iy 5 Y2 0 2 Advanced Options E Manhattan Plot 7 Q Q Plot Line SNP Position Chromosome Cut off Value 1 0E 11 15 Threshold Value Line 1 0E 26 37 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 i Rn E Save Graph s h gt 7 CN AR m Oy Chromosome 1 Mb logi10 1 p o co urb vw anyer PUIBLIO Significance Figure 12 Effect setting dialog for selecting only a few effects upper and chromosome graph with only a few effects lower 29 Effects Tota 31 Y1 Axis amp logl10 1 p Y2 Axis logt t p Original
26. P All Traits TRAITO0 10 I1 i1 p0 P1 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome Cut off P value Threshold P value Line 4e 12 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Rn 00 Save Graph s Results SNP All Traits TRAITOO ManhattanPlot l1 p1 P1 90 Results SNP All Traits TRAITOO Q QPlot Results SNP All Traits TRAITOO 1 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 2 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 3 I1 i1 p1 P1 g0 Results SNP All Traits TRAITO0 4 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 5 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 6 I1 i1 p1 P1 90 Results SNP All Traits TRAITOO 7 11 i1 p1 P1 g0 Results SNP All Traits TRAITOO 8 11 i1 p1 P1 g0 Results SNP AII Traits TRAITOO 9 11 i1 p1 P1 g0 Results SNP AII Traits TRAITOO 10 11 i1 p1 P1 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome I IN Il i Cut off P value 4e 7 Threshold P value Line 4e 12 i eo y e 12 68 10 59 E Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 11 40 LJ N 1 t al x t L4 d p m L4 pr ry L4 v ro g L4 5 P Save Graph s
27. P21 90 Results MixedValues All 9 I1 i0 p10 p20 P11 P21 90 Results MixedValues All 10 I1 10 p10 p20 P11 P21 90 Results MixedValues All 11 I1 10 p10 p20 P11 P21 g0 Results MixedValues All 12 I1 10 p10 p20 P11 P21 g0 Results MixedValues All 13 I1 10 p10 p20 P11 P21 g0 4 m Input File IPEVG Results MixedValues All snpe Effect s Tota 31 YA 16 Y2 0 15 Advanced Options E Manhattan Plot gt E Q Q Plot Line v SNP Position anyer PUIBLIO Q T e T C 2 I q e i a 2 a c Un Chromosome Cut off Value V Threshold Value Line 1 0E 26 37 Shade Points below Threshold Line Line needs to be off v Unknown Chromosome 32 Run Save Graph s Current Graph All Graphs Figure 10 Chromosome graph including threshold value lines for both axes 4 3 Chromosome Graphs Graphs may be drawn for all chromosomes Figure 9 Each chromosome graph has the options to use or not to use lines connecting data points as shown in the upper and lower figures in Figure 9 Genetic effects or original values can be displayed by Y1 or Y axis In Figure 10 16 genetic effects are displayed by Y 1 axis using log10 1 0 value and 15 values are displayed by Y axis using original values The legend displays the 16 genetic effects in the left three columns and the 15 original values in the right two column
28. T1 or T2 in this example Any type of user defined genetic effect could be included as a value column For example one type of genetic effect such as additive effects for all traits could be placed in one input file as shown in the following example named SNPEVG2 _ input2 snpe SNPID Chr Position Tl add T2 add T3 add T4 add T5 add SNPO1 1 571340 2 48E 03 1 11E 03 1 58E 03 2 74E 02 2 65E 01 SNPO2 1 845494 7 02E 04 9 07E 04 8 91E 05 2 56E 02 8 02E 02 SNPO3 1 883895 1 87E 02 1 71E 01 3 07E 03 1 07E 02 2 81E 03 SNPOA 1 929617 1 79E 03 1 57E 702 1 39E 05 9 03E 02 2 99E 04 SNPO5 I 950841 1 20E 03 7 07E 03 1 32E 05 8 46E 02 1 71E 03 17 SNPEVG2 supports viewing and graphing original values for general use in addition to genetic effects In the following input file named SNPEVG2 input3 snpe one type of genetic effect for 3 traits is included from column 4 through column 6 and two different original values are included in column 7 and column 8 SNPID Chr Position Tl add T2 add T3 add G1 G2 SNPO1 1 571340 2 48E 03 1 11E 03 1 58E 03 1 74E 00 6 35E 02 SNPO2 1 845494 7 02E 04 9 07E 04 8 91E 05 3 52E 01 2 34E 00 SNPO3 1 883895 1 87E 02 1 71E 01 3 07E 03 1 73E 02 2 81E 01 SNPOA T 929617 1 79E 03 ds 7E 02 1 39E 05 7 53E 01 4 49E 04 SNPO5 1 950841 1 20E 03 7 07E 03 1 32E 05 5 73E 01 1 11E 01 For the given first two input files SNPEVG2 inputl snpe and SNPEVG2 input2 snpe SNPEVG2 plots the log10 1 p values according to the SNP positions on ea
29. Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md Input File C SNPEVG single locus fig out SNP Position C SNPEVGIsnp postionpos SNP Effects Genotypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options 7 Manhattan Plot 3 Q Q Plot Line OTa E Chromosome E Cut off P value E Threshold P value Line pal tfh SNK e tS Shade Points below Threshold Line Line needs to be off V Unknown Chromosome 32 E Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Save Graph s EC Xem AS e wheal Genotypic Significance Q Q Plot Traiti single_locus_fig Traitl ManhattanPlot l0 p0 P0 g0 mg single_locus_fig Trait ManhattanPlot l0 p0 P0 g0 ma single_locus_fig Traitl ManhattanPlot 10 p0 P0 g0 md single_locus_fig Traitl Q QPlot mg single_locus_fig Traitl Q QPlot ma single_locus_fig Traitl Q QPlot md Input File C SNPEVG single_locus_fig out SNP Position C SNPEVG snp position pos SNP Effects Genotypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options 7 Manhattan Plot 3 Q Q Plot Line El Chromosome E Trait El Cut off P value E Threshold P value Line Shade Points below Threshold Line Line
30. Value wa ME erect EE eom IE eer m E E a E GVALUEOS tvm ems FI A FTE IP em mg FT E n MEE enne E FI I F E CVALUEOS F E GVALUEO e xS 55 x5 3 3 3 3 JE C GVALUEI0 v2 cen E n cue B n crrectis e me Gaia s L LIGVALUE11 X GVALUE13 Results MixedValues All 1 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 2 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 3 I1 10 p11 p21 P11 P21 90 Results MixedValues All 4 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 5 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 6 I1 10 p11 p21 P11 P21 90 Results MixedValues All 7 I1 10 p11 p21 P11 P21 90 Results MixedValues All 8 I1 i0 p11 p21 P11 P21 90 Results MixedValues AlI 9 I1 10 p11 p21 P11 P21 90 Results MixedValues All 10 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 11 I1 10 p11 p21 P11 P21 90 Results MixedValues All 12 11 10 p11 p21 P11 P21 90 Results MixedValues All 13 11 10 p11 p21 P11 P21 g0 Input File IPEVG Results MixedValues All snpe Effect s Total 31 Yi 5 Y2 0 2 Advanced Options El Manhattan Plot 3 E Q Q Plot E Line SNP Position E Chromosome V Cut off Value 10E M 15 V Threshold Value Line 10E 26 37 E Shade Points below Threshold Line Line needs to be off v Unknown Chromosome 32 Save Graph s Chromosome 1 Mb o Weer ee Significance log10 1 p anyer PUIBLIO
31. at provides a wide range of options for the user to choose The GUI can produce the Manhattan plot 1 the Q Q plot 2 of all SNP effects and graphs for SNP effects by chromosome by clicking one command Any or all the graphs can be saved as high resolution png files by clicking one command The user can also increase the png resolution by using full screen For example the default screen size give a 667x667 png resolution and full screen gives 1119x1119 for a 1900x1200 screen resolution The program allows users to select traits for graphing and viewing The user also has the options to draw graphs using lines or symbols or specify a cut off p value to be shown on each graph or threshold p value line for SNP effects to be included in the graphs The main features and options of SNPEVGI can be summarized as follows e Instant graphics of large quantities of genome wide significance testing results from any methods for any or all traits e Input format conversion from output file of any GWAS software to the input file format for SNPEVG e Immediate viewing of all graphics within each SNPEVGI GUI e Scalable SNPEVGI GUI allowing efficient and flexible use of computer screen e Graphics of Manhattan plot Q Q plot and P value figures for all chromosomes and all traits up to 100 traits by one click of run e Save of one graph all graphs for all chromosomes of a trait or all graphs for all chromosomes and all traits up to 100 traits by one of
32. attan Plot Q Q Plot Line SNP Position Chromosome Cut off P value Threshold P value Line 4e 10 Shade Points below Threshold Line Line needs to be off W Unknown Chromosome 32 Save Graph s Chromosome Current Graph Current Trait ta cm n ete te Yu Par b Bees eden pss i Cr pos o Fe UHR 1 T mt he CM Ey ZEAE AE II e LJ m WA eaa Dt Mes ael o Jy yew e 2A 1 age a am ls wate C f S eT tane c meet Le y We nik gt CEG tsa wo os seww e s t trash h ee ENT AS en E uz SAE tu cer est C JP er le Lind 9 at won t t fusci ye s parois D prv 3 be bat t V uc nm aA on Figure 6 Manhattan plot with shading of data points below threshold P value line Basic colors T Sse EEE Dynamic size Chromosome Color C Template 1 L ia Template 2 ia L LI Template 3 Lj L Template 4 L L LI eal Template 5 ia L ia LJ r gt SNP Effect View and Gri TRAITOO v Results SNP All Traits TRAITOO ManhattanPlotll plPl gl Results SNP All Traits TRAITOO Q QPlot Results SNP All Traits TRAITOO 1 11 i0 p1 P1 g1 Results SNP All Traits TRAITOO 2 I1 i0 p1 P1 g1 Results SNP All Traits TRAITO0 3 11 i0 p1 P1 g1 E Results_SNP_All_Traits TRAITOO 4 IL i0 p1 P1 g1
33. cal tool for SNP effect viewing and graphing The program currently uses the output file of single locus test results from the epiSNP computer package 1 as the input file for drawing figures Each chromosome figure can display three genetic effects genotypic additive and dominance effects and the number of observations Alternatively the user may follow the file format of the default input file for graphical viewing and drawing For example the user could replace the four columns of genotypic effect additive effect dominance effect and the number of observations by four columns of additive effects from four different traits 6 INSTALLATION 6 1 Windows x86 x64 The installation of SNPEVG3 only requires to create a folder to place all files in SNPEVG zip in that folder and then change SNPEVG3 exe file to SNPEVG3 exe file The Windows x64 64 bit version 1s available by request 6 2 Mac OS X 10 6 or newer 64 bit The installation of SNPEVG3 only requires to unzip SNPEVG3 Mac zip file to obtain SNPEVG3 app file 7 FILE FORMATS One input file with p values in log10 1 p scale is required An example is the output file from the epiSNP package named single locus fig out The following is an example of this file The output for making figures m a d quest Chr Locus M A D Ind traiti 14 M112 0 704E 00 0 107E 01 0 285E 00 295 trait2 14 M112 0 492E 00 0 127E 00 0 864E 00 295 ead 14 M212 0 401E 00 0 689E 01 0 748E 00 295 traitl 19
34. ch chromosome for SNP markers with known chromosome positions or in sequential orders of the SNP markers for SNPs in the chromosome file specified as unknown by the user For the given third input file SNPEVG2 input3 snpe SNPEVG2 is capable of plotting the first three value columns genetic effects by log10 1 p values and plotting the last two value columns by original values For each column SNPEVG2 produces the following graphs e Manhattan plot with options For each column of genetic effects SNPEVG2 produces the following graphs e Q Q plot with one option For all columns of genetic effects and original values SNPEVG2 produces the following graph e Graphs for one or all chromosomes with options 4 RUNNING SNPEVG2 To run SNPEVG2 open the folder that holds the SNPEVG2 files and required input files Double click SNPEVG2 exe For Mac users double click SNPEVG2 app will open the GUI of SNPEVG2 as shown in Figure 1 below Figures in the manual are produced in Windows 7 The GUI shown below is scalable 1 e the GUI size can be changed by dragging the borders of the screen so that the GUI can use the full screen or part of the screen allowing efficient and flexible use of the computer screen 4 1 Upload Input File Select Options and Run In the GUI of Figure 1 upload an input file by clicking Browse select the options and then click Run to produce the corresponding graphs Alternat
35. esting or original values from any methods up to 100 effects values The chromosome figure also supports two axes Y1 and Y2 and the genetic effects or original values can be designated to either Y 1 or Y axis for two groups of scales SNPEVG2 provides a wide range of options for the user to choose The GUI can produce the Manhattan plot 1 the Q Q plot 2 and graphs for all values by chromosome by clicking one command Any or all the graphs can be saved as png files by clicking one command The user can increase the png resolution by using full screen For example the default screen size give a 667x667 png resolution and full screen gives 1119x1119 for a 1900x1200 screen resolution The program allows users to select values from different effects or test methods for graphing and viewing The user also has the options to draw graphs using lines or symbols or specify a cut off value to be shown on each graph or threshold value line to be included in the graphs The main features and options of SNPEVG2 can be summarized as follows e Instant graphics of large quantities of genome wide significance testing results or original values from any selected significance effects and original values e Support of Y1 and Y axes and selection of values for either axis e Immediate viewing of all graphics within the SNPEVG2 GUI e Scalable SNPEVG2 GUI allowing efficient and flexible use of computer screen e Graphics of Manhattan plo
36. etermined by the user and are specified in the parameter file This intput file must contain the SNP marker chromosome position and at least one p value for producing the output SNPE file 4 RUNNING SNPEVGconvert 4 1 Windows To run SNPEVGconvert one executable file parameter dat and the input GWAS file must be in the same directory The program can be run in two ways Method 1 1 Open computer and click the folder where the SNPEVGconvert executable file and its input files are located 2 Double click the executable file e g SNPEVGconvert exe Method 2 Open the C prompt window and type gt chdir c SNPEVOGconvert location gt SNPEVGconvert executable file e g SNPEVGconvert exe 4 2 Mac OS X 64 bit To run SNPEVGconvert one executable file parameter dat and the input GWAS file must be in the same directory The program can be run in the following way Method 1 Open a terminal and type gt cd SNPEVGconvert location gt SNPEVGconvert executable file e g SNPEV Gconvert 34 Part IV SNPEVG3 Three effects and sample size per trait EPI SNP output file as input file Contents EB EU DN ION merrnin A A A AA 3 INSTALLATION sesessssseessssscsssscessssccosssscesssecosssecosssecossscctosssceosseeeosssceossscesssscessseeossseeossseosssstosssecosssetessseeess TEL SUR Em d OBUNNINGSNPEVCS Pe HEU TREHRERUIERHICUEHNTIORU eC ORE ES EET IR EDU 35 5 INTRODUCTION SNPEVG3 is a graphi
37. etic effects and original values by chromosome with lines upper and with symbols lower GVALUEOS T GVALUEO9 GVALUE10 OGVALUE11 GVALUE12 x GVALUE13 Y GVALUE14 26 anyer EulDLIO anyer euiDLIO Results MixedValues All 1 I1 i1 p10 p20 P10 P20 90 Results_MixedValues_All 2 11 i1 p10 p20 P10 P20 g0 Results_MixedValues_All 3 11 i1 p10 p20 P10 P20 g0 Results MixedValues All 4 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 5 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 6 I1 i1 p10 p20 P10 P20 g0 Results MixedValues All 7 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 8 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 9 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 10 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 11 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 12 I1 i1 p10 p20 P10 P20 90 Results MixedValues All 13 I1 11 p10 p20 P10 P 20 90 4 Input File IPEVG Results_MixedValues_All snpe Effect s Tota 31 Yi 16 Y2 0 15 Advanced Options Manhattan Plot E Q Q Plot Line SNP Position Chromosome E Cut off Value E Threshold Value Line i Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Ren 0 Save Graph s Results MixedValues All 1 I1 i0 p10 p20 P10 P20 Results MixedValues All 2 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 3 I1 i0 p10 p20 P10 P20 90 Results MixedValues All 4 I1 i0 p10 p20 P10 P20 90
38. f u c RDM om ot oof E oft 3508 ge TIST prepsat os ef s 2 8 EXIT PL UM ERE RH uut eet o rer a eit s det So va Petes loe omnia er Te lt Hed or e We pa ide r M ine put vete o H p SS eterna 9 Tie bat fin wv ut a kr 7 zo gt IAEA a ABl r apum wee e y Chromosome s e E w tes L MR lt r Sst sian e os v to s s rut T oat n a n soo d MW ate NL Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Save Graph s E Figure 5 Manhattan plot without showing data points below cut off value Results MixedValues All ManhattanPlot EFFECTOO I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOZ1 I1 p10 p20 P E Results MixedValues All ManhattanPlot EFFECTO2 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO3 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO04 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOS I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO6 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO7 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO8 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO9 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT10 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT11 I1 p10 p20 P
39. ff P value is specified the Q Q plot only displays P values above the cut off P value We do not recommend this option for Q Q plot A SNP Effect View and Grap TRAITOO Q Q Plot TRAITOO Results SNP All Traits TRAITOO ManhattanPlot I1 pO P0 g0 Results SNP All Traits TRAIT00 Q QPlot n Results_SNP_AIl_Traits TRAITOO 1 11 i1 p0 P0 g0 Results_SNP_AIl_Traits TRAITOO 2 11 i1 p0 P0 g0 Results_SNP_AIl_Traits TRAITOO 3 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 4 11 i1 p0 P0 g0 Results SNP AII Traits TRAITOO 5 I1 i1 p0 P0 g0 Results SNP All Traits TRAITOO 6 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 7 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 8 I1 i1 pO PO g0 Results SNP All Traits TRAITOO 9 I1 i1 p0 P0 g0 Results SNP AII Traits TRAITOO 10 I1 i1 p0 P0 g0 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options v Manhattan Plot gt 7 Q Q Plot V Line V SNP Position L Chromosome C Cut off P value E Threshold P value Line Shade Points below Threshold Line Line needs to be off v Unknown Chromosome 32 Run Save Graph s Expected Significance log10 1 p Current Graph Current Trait All Graphs 0 00 2 73 5 47 8 20 10 93 13 67 1640 19 14 21 87 24 60 27 34 Figure 8 Q Q plot of all SNP effects for each trait 4 4 Chromosome Graphs Graphs may be drawn for all chromosome
40. gle locus and epistatic SNP effects of quantitative traits in genome wide association studies BMC Bioinformatics 2008 9 315 323 2 Zhao JH gap Genetic analysis package J Stat Softw 2007 23 108 44
41. hattanPlot EFFECTO09 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT10 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT11 I1 p11 p20 P Results MixedValues All ManhattanPlot EFFECT12 I1 p11 p20 P 4 m n QUAM ge i t Input File IPEVG Results MixedValues All snpe Effect s Total 31 Y1 16 Y2 0 15 Advanced Options v Manhattan Plot gt Q Q Plot Line IV SNP Position ee e v Ll I y A Po T 9i ut egt 4 2 en e KT LN 2 m ert pss 5 o D v LII rs J os o eti t P we DARI Race 7 Significance log10 1 p etn of de H Im o tuo ut t LI Chromosome Cut off Value 1 0E 10 Threshold Value Line 4 0E 23 J Shade Points below Threshold Line Line needs to be off v Unknown Chromosome 32 5 ee loa e ee mat gets Aem rs ime qe LO 3 pet ut ho s a z TERT Mat irad ph Lu ae ev E A a epu m P aan ke p on me p eg etes fe f ve VU P 3 etate n HA A my dent Meme des Save Graph s Current Graph All Graphs Figure 7 Manhattan plot with user s color selection upper and Manhattan plot with cut off value and shading of data points below threshold value line lower In the Manhattan plot each chromosome width is the true width of
42. ified the Q Q plot only displays P values above the cut off P value We do not recommend this option for Q Q plot 40 Genotypic Significance Manhattan Plot Traiti single_locus_fig Traitl ManhattanPlot I1 p0 P1 gl mg single locus fig Traitl ManhattanPlot I1 p0 P1 g1 ma single locus fig Traitl ManhattanPlot I1 p0 P1 g1 md single locus fig Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md Input File C SNPEVG single locus fig out SNP Position C SNPEVG snp_position pos SNP Effects Genotypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options Manhattan Plot Q Q Plot Line Chromosome E Trait Cut off P value Threshold P value Line 4E 10 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 392 Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Lo Rn 0 jJ Save Graph s Genotypic Significance Manhattan Plot Traiti single locus fig Traitl ManhattanPlot I1 p1 P1 91 mg single locus fig Traitl ManhattanPlot I1 p1 P1 g1 ma single locus fig Traitl ManhattanPlot I1 p1 P1 91 md single locus fig Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md Input File C SNPEVG single locus fig out SNP Position C SNPEVG snp_position pos SNP Effects Genot
43. ig Trait1 01 10 i1 e0 p0 P0 g0 sa single locus fig Trait1 02 10 i1 e0 p0 P0 g0 sa single locus fig Trait1 03 10 i1 e0 p0 P0 g0 sa single locus fig Trait1 04 10 i1 e0 p0 P0 90 sa single locus fig Trait1 05 10 i1 e0 p0 P0 90 sa single locus fig Trait1 06 10 i1 e0 p0 P0 g0 sa Input File C SNPEVG single locus fig out SNP Position C SNPEVG snp_position pos SNP Effects Genotypic Significance Dominance Significance V Additive Significance Number of Observations Advanced Options 7 Manhattan Plot gt E Q Q Plot v Line E Chromosome E Trait Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off V Unknown Chromosome 32 4 Sort SNP Position needs to be off D Genotypic Significance O Dominance Significance Additive Significance Number of Observations Run uo id s Pd O Nf 1 ry oy We NY IM NS Sw Kf se Pi te d e y gt VU e a QN Save Graph s g Es cy e a f eX P y wa 49 qe sf A QM P NC X w S Clfrom som LS P qv g Current Graph Current Trait All Graphs g Q a g E vou g J x i s F x i d y ex VP Figure 7 SNP effects are sorted according to the significance of one effect given that the position option is turned off 4 2 Chromosome Graphs by SNPEVG3 By default each chromosome graph by SNPEVG3 displays three genetic effects
44. ions V Manhattan Plot gt 7 Q Q Plot Line Chromosome Trait Cut off P value V Threshold P value Line 0 0000000004 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Save Graph s Chromosome Current Graph Current Trait All Graphs Figure 3 Manhattan plot setting for pixel sizes and chromosome colors upper and Manhattan plot with threshold P value line 39 Genotypic Significance Trait1 Manhattan Plot Traiti single locus fig Traitl ManhattanPlot I1 p1 P0 g0 mg single locus fig Traitl ManhattanPlot I1 p1 P0 g0 ma single locus fig Traitl ManhattanPlot I1 p1 P0 g0 md single locus fig Traitl Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md Input File C SNPEVG single locus fig out Browse V SNP Position C SNPEVG snp_position pos SNP Effects V Genotypic Significance 7 Dominance Significance V Additive Significance Number of Observations Advanced Options 7 Manhattan Plot gt 7 Q Q Plot IV Line L Chromosome Trait V Cut off P value 0 0000004 Threshold P value Line Shade Points below Threshold Line Line needs to be off V Unknown Chromosome 32 C S
45. ively the user can drag the SNPE input file and drop to the input field without using browse button This program supports maximum 100 value columns for graphics display and the extra value columns in the input file are discarded When the input file 1s specified total number of value columns and number of selected value columns are displayed as shown in the upper figure in Figure 2 All 31 value columns are selected and are displayed for axis Y1 by default Clicking Setting a dialog in 18 the lower left figure in Figure 2 pops up and allows the user to select the values that will be displayed The user can change each value setting for the axis Y1 or Y by single clicking the symbol of Y1 or Y2 and the setting toggles between the symbol Y1 and Y2 The user can also select either axis to be the log10 1 p value or the original value and change value texts for the display of the legend The number of total value columns and the number of selected value columns for Y1 or Y2 are displayed and updated according to the change of the setting If the axis is specified with the log10 1 p effect Il 1s displayed besides the axis notation of Y1 or Y2 If the axis is specified with the original value o is displayed instead In addition the symbol color for each value can be changed by double clicking the region of a color and selecting a new color from color selection dialog The user can also drag to co
46. l 2 IL i0 p11 p21 P11 P21 g0 Results MixedValues All 3 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 4 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 5 I1 i0 p11 p21 P11 P21 90 T Results MixedValues All 6 I1 iQ p11 p21 P11 P21 0 Results MixedValues All 7 I1 10 p11 p21 P11 P21 90 Results MixedValues All 8 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 9 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 10 I1 10 p11 p21 P11 P21 90 Results MixedValues All 11 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 12 I1 i0 p11 p21 P11 P21 90 Results MixedValues All 13 I1 i0 p11 p21 P11 P21 90 4 Input File IPEVG Results MixedValues All snpe Effect s Tota 31 Yt 16 Y2 oy 15 Advanced Options 7 Manhattan Plot 7 Q Q Plot Line SNP Position Chromosome Cut off Value 1 0E 11 15 Threshold Value Line 1 0E 26 37 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Lo Rn SE Save Graph s Significance log10 1 p anyer euiDLIO Chromosome 2 Mb Figure 11 Chromosome graph with threshold value lines and cut off values for chromosome 1 upper and chromosome 2 lower 28 Effects Tota 31 Yi Y1 Axis log10 1 p Original Value Y2 Axis logt t p Origir ma 7 Ful M I IE EFFECTUS Ima lerrectos pr ES eFrectos Pa eFrectos SVALUETI v2 IPSE I E eves nouo x x3 3 3 E s es Les es Les
47. of SNPEVGI The GUI shown above is scalable 1 e the GUI size can be changed by dragging the borders of the screen so that the GUI can use the full screen or part of the screen allowing efficient and flexible use of the computer screen 555 Trait Selection Traits Tota 31 TRAITOO W TRATO MW TRAITO4 TRATOS w TRATOS TRATOT v TRATOS W TRATOS v TRAIT10 v TRAIT11 v TRAIT12 v TRAIT13 v TRAIT14 4 TRAIT15 Selected 31 TRAIT18 v TRAIT17 W TRAIT18 v TRAIT19 W TRAIT20 v TRAIT21 v TRAIT22 v TRAIT23 v TRAIT24 W TRAIT26 v TRAIT27 W TRAIT28 v TRAIT28 Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome C Cut off P value E Threshold P value Line Shade Points below Threshold Line Line needs to be off E Unknown Chromosome Save Graph s Guren Tan Figure 2 Upload of an input file upper and selection of traits to display lower 4 1 Upload Input File Select Options and Run In the GUI of Figure 1 upload an input file by clicking Browse select the options and then click Run to produce the corresponding graphs Alternatively the user can drag the SNPE input file and drop to the input field without using browse button This program sup
48. ort SNP Position needs to be off 3 Genotypic Significance Dominance Significance Additive Significance Number of Observations Run Save Graph s Chromosome Current Graph Current Trait All Graphs Figure 4 Manhattan plot showing data points above cut off P value 4 1 Manhattan and Q Q Plots The Manhattan plot 2 and Q Q plot 3 by SNPEVG3 have the same features as described for SNPEVGI except the figure legend for the Manhattan plot Figure 2 The Manhattan plot has the options to change the setting of pixel sizes and colors by clicking the icon beside its checkbox Figure 3 The setting of pixel sizes has two options The fixed pixel size allows the user to change the size of pixels between 1 and 9 The dynamic pixel size allows the pixels to be varied according to the data points significance within the user defined range The color of each chromosome in Manhattan plot is assigned according to the selected color template The template colors are extended by rotation and can be changed by double clicking the region of a color to select a new color or moving a color by dragging and dropping the color region in the same template Manhattan plot also has options to mark the threshold P value line for declaring significance Figure 3 to show data points above the cut off P value Figure 4 or to shade data points below threshold P value line Figure 5 Warning If a cut off P value is spec
49. place all files in SNPEVG zip in that folder and then change SNPEVG2 exe file to SNPEVG2 exe file The Windows x64 64 bit version 1s available by request 2 2 Mac OS X 10 6 or newer 64 bit The installation of SNPEVG2 only requires to unzip SNPEVG2 Mac zip file to obtain SNPEVG2 app file 3 INPUT FILE FORMATS One input file with the extension snpe for p values or original values is required The format of the input file 1s Column 1 SNP name Column 2 chromosome number Column 3 SNP position on the chromosome Columns 4 through Column n3 value columns of P values or original values for total n effects and values where 0 lt n lt 100 As an example the following is an input file named SNPEVG2 inputl snpe SNPID Chr Position Tl geno Tadd T1 dom T2 geno T2 add T2 dom SNPO1 L 571340 2449E 03 Te TERSUS looi 2 74E 02 ZoGOBEDUI Jg SNPO2 js 845494 1402 1204 92 0 ZE QA Ou o ESQ 2 902702 O UZER UZ OJGDTER DOd SNPO3 al 0238S L gS OZ ler lE 34 0 LE 03 l0 7E 92 ZLE IzTIESOI SNPO4 1 929617 Te RISUS lu 5g02 49 3 9 023E 02 zu mds La I A SNPO5 al 950841 lw 20E 0 T 07E 03 DOORS 9 426E 02 lio4LPECDO 207E D3 In the above file column 1 1s the marker name column 2 1s the chromosome number column 3 is the marker position in units of base pairs bp or genetic distance in centi Morgans and column 4 through column 9 are P values of genotypic effect geno additive effect add and dominance effect dom for each trait
50. ports maximum 100 traits for graphics display and the extra traits in the input file are discarded When the input file is specified total number of traits and number of selected traits are displayed as shown in the upper figure in Figure 2 All traits are selected by default Clicking Select a dialog in the lower figure in Figure 2 pops up and allows the user to select the traits that will be displayed 4 2 Manhattan Plot The Manhattan plot allows all SNP effects of all chromosomes per trait In the GUI of the upper figure in Figure 2 check Manhattan Plot The program will produce a Manhattan plot a Q Q plot and chromosome graphs for each trait column BE SNP Effect View and Grog TRAITOO Manhattan Plot TRAITOO Results SNP All Traits TRAITOO ManhattanPlot I1 p0 P0 g0 Results SNP All Traits TRAIT00 Q QPlot Results SNP All Traits TRAITOO 1 I1 i1 p0 P0 g0 Results SNP All Traits TRAITOO 2 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 3 11 i1 p0 P0 g0 Results SNP All Traits TRAITO0 4 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 5 I1 i1 p0 P0 g0 Results SNP All Traits TRAIT00 6 I1 i1 p0 P0 g0 Results SNP All Traits TRAITOO 7 I1 i1 p0 P0 g0 Results SNP All Traits TRAITOO 8 I1 i1 p0 P0 g0 Results SNP All Traits TRAITOO 9 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 10 I1 i1 pO P0 gO Input File NPEVG Results SNP All Traits snpe Traits Total 31 Display 31 Advanced Options
51. py a symbol color and drop to change another symbol color 4 2 Manhattan Plot The Manhattan plot allows all SNP effects or original values of all chromosomes per value column In the GUI of the upper figure in Figure 2 Manhattan Plot is enabled by default The program produces Manhattan plots and chromosome graphs for each value column The left window of SNPEVG2 in Figure 3 is the Graph window for viewing graphs The Manhattan plot is always the first graph in the list of each genetic effect or original value s graphs and the first graph 1s the default display of all graphs SNP Effect View and Graph Input File Browse Effect s Total Y1C Y2 Advanced Options W Manhattan Plot 3 7 Q Q Plot Line J SNP Position Chromosome Cut off Value Threshold Value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome Save Graph s Current Graph All Graphs Figure 1 Graphical User Interface GUI of SNPEVG2 19 Input File IPEVG Results MixedValues All snpe Effect s Total 31 Yi 31 Y2 0 0 Advanced Options Manhattan Plot Q Q Plot C Line SNP Position E Chromosome E Cut off Value E Threshold Value Line M C Shade Points below Threshold Line Line needs to be off E Unknown Chromosome Save Graph s Tota 31 Yt Y1 Axis log10 1 p Y2 Axis
52. s Figure 9 or one selected chromosome To select a chromosome the specified chromosome name must match the name in the input file Each chromosome graph has the options to use or not to use lines connecting data points Figure 9 to use a threshold P value line Figure 10 or display data points above the cut off P value as shown in the lower figure in Figure 10 11 Results SNP All Traits TRAITOO ManhattanPlot l1 pO P0 g0 Results SNP All Traits TRAITOO Q QPlot Results SNP All Traits TRAITOO 1 I1 i1 p0 PO g0 Results SNP All Traits TRAITOO 2 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 3 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 4 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 5 I1 i1 p0 P0 g0 Results SNP All Traits TRAITO0 6 11 i1 p0 P0 g0 Results SNP All Traits TRAITOO 7 l1 i1 p0 P0 gO Results SNP AII Traits TRAITOO 8 I1 i1 p0 P0 gO Results SNP AII Traits TRAITOO 9 11 i1 p0 P0 g0 Results SNP AII Traits TRAITOO 10 11 i1 p0 P0 g0 Input File NPEVG Results SNP All Traits snpe Traits Tota 31 Display 34 Select Advanced Options Manhattan Plot Q Q Plot Line SNP Position Chromosome Cut off P value Threshold P value Line Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Rn 0 Save Graph s Results SNP All Traits TRAITOO ManhattanPlot l1 p0 P0 g0 Results SNP All Traits TRAITOO Q QPlot Results SNP All Traits TRAITOO 1 I1 i0 pO P0
53. s separately The number of columns in the legend is automatically adjusted when the application window is resized Threshold value lines for either Y1 or Y axis or both axes can be included Figure 10 The program can display data points above the cut off value and discard other points for chromosome 1 in the upper figure in Figure 11 and switch to chromosome 2 by down arrow key in the lower figure in Figure 11 The user can display only one chromosome graph by specifying the chromosome name To select a chromosome the specified chromosome name must match the name in the input file The user can select only a few values to be displayed in the effect setting dialog as shown in the upper figure in Figure 12 The lower figure in Figure 12 shows that 5 effects and 2 original values are displayed for the axis Y1 and Y respectively The program allows the user to copy a color between effects or values in effect setting dialog by dragging a color from one color region to another The upper figure in Figure 13 shows that the blue color is replicated from one effect to all others for Y1 axis and the purple color is replicated for two original values of Y axis The lower figure in Figure 13 shows the graph of using blue color for 5 selected effects of Y 1 axis and using purple color for 2 selected original values of Y axis zy log10 1 p Significance Oo cr rb v X GVALUEOO V GVALUEO1 A GVALUEO2
54. t ee ee ee eter ed p t e 99 gee Rae S 2 E pr e NR n B Ve ahi eva oT at wee ERE PLE p gy a T9 Rites e 00 ete a M ed La 3 Muck Geek ee Wek om eo ff g ro ES e Matre s Tt 9 9 n p co Seana cle aoe M on Seige ve ce t fet we TYIY 4 aw SSO S si Utt hans ar m i Figure 5 Manhattan plot with threshold P value line and without showing data points below cut off P value Manhattan Plot TRAITOO Results SNP All Traits TRAITOO ManhattanPlot Il p0 P1 g1 Results_SNP_AIl_Traits TRAIT00 Q QPlot Results_SNP_AIl_Traits TRAIT00 1 11 i0 p0 P1 g1 Results SNP All Traits TRAITOO 2 11 10 p0 P1 g1 Results SNP All Traits TRAITOO 3 11 10 p0 P1 g1 Results SNP All Traits TRAITOO 4 11 10 p0 P1 g1 Results SNP All Traits TRAIT0O 5 I1 i0 pO P1 91 Results SNP All Traits TRAITOO 6 I1 i0 pO P1 g1 Results SNP All Traits TRAITOO 7 11 10 p0 P1 g1 Results SNP All Traits TRAITOO 8 I1 i0 pO P1 g1 Results SNP All Traits TRAIT00 9 11 10 p0 P1 g1 Results SNP All Traits TRAITOO 10 I1 i0 pO P1 g1 Input File NPEVG Results SNP All Traits snpe Browse oon s e c tet et ye eA on wA VERE et t s 1 49 canes GU fee 9 a I a cape ee e o t Traits Total 31 Display 31 Advanced Options Manh
55. t Q Q plot and figures for all chromosomes and all effects values up to 100 effects values by one click of run e Save of one graph or all graphs for all Manhattan plots Q Q plots and chromosomes by one of Current Graph or All Graphs e Each Manhattan plot has the following options and features Each chromosome width 1s the true width of the chromosome defined by the starting and ending SNP marker positions Adjustable fixed pixel size and dynamic pixel size Color templates with the capability of custom colors Horizontal line marking the threshold value for declaring significance Display of values above the specified cut off value Shading values below the threshold value line e Each chromosomal figure of all values has the following options Selection of values to be included in the figure One or two groups of values for Y1 or Y2 axes log 1 p vs original value Horizontal line marking the threshold value for declaring significance Display of values above the specified cut off value Shading values below the threshold value line Optional use of lines to connect data points User specified unknown chromosome that will plot values in the sequential order of SNP markers rather than chromosome positions e Q Q plot for each significant effect displaying all data points 16 2 INSTALLATION 2 1 Windows x86 x64 The installation of SNPEVG2 only requires to create a folder to
56. t EFFECTO03 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO4 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOS I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO6 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTO07 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOS8 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTOO9 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECTI10 I1 p10 p20 P e eh ce Results MixedValues All ManhattanPlot EFFECTI11 I1 p10 p20 P Results MixedValues All ManhattanPlot EFFECT12 I1 p10 p20 P 4 m p a e c Input File IPEVG Results MixedValues All snpe Effect s Total 31 s o sees ee e 9 T c p j 25 9 i zi ele uL we st wv ook nee Advanced Options v Manhattan Plot 3 7 Q Q Plot Line V SNP Position Ya epe PF v9 1 v Significance log10 1 p Ar eJ RP Chromosome C Cut off Value V Threshold Value Line 4 0E 23 Shade Points below Threshold Line Line needs to be off v Unknown Chromosome 32 wo l Run Save Graph s Chromosome Current Graph All Graphs Figure 4 Manhattan plot setting for pixel sizes and chromosome colors upper and Manhattan plot with threshold value line lower 22 Q T L Ce T U 2 I oq e C qm 2 t D Un tX e Fhe 994 puo o 4 8 oF
57. tanPlot GVALUE11 I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUE12 I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUE13 I1 p10 p20 F Results MixedValues All ManhattanPlot GVALUE14 I1 p10 p20 F Results MixedValues All Q QPlot EFFECTOO Results MixedValues All Q QPlot EFFECTO1 Results MixedValues All Q QPlot EFFECTO2 Results MixedValues All Q QPlot EFFECTO3 Results MixedValues All Q QPlot EFFECTO4 Results MixedValues All Q QPlot EFFECTO5 Results MixedValues All Q QPlot EFFECT06 Results MixedValues All Q QPlot EFFECTO7 Results MixedValues All Q QPlot EFFECTOS 4 Tm Input File IPEVG Results MixedValues All snpe Effect s Total 31 Y1 16 Y2 0 15 Advanced Options v Manhattan Plot v Q Q Plot Line SNP Position Observed Significance log10 1 p Chromosome F Cut off Value Threshold Value Line Shade Points below Threshold Line Line needs to be off V Unknown Chromosome 32 Save Graph s Expected Significance log10 1 p Current Graph AN Graphs 0 00 2 73 5 47 8 20 10 93 13 67 16 40 19 14 21 87 24 60 27 34 Figure 8 Q Q plot of all SNP effects for each genetic effect 25 log10 1 p Significance Chromosome 1 Mb Significance log10 1 p X GVALUEOO V GVALUEO1 A GVALUEO2 GVALUE03 GVALUE04 GVALUEOS O GVALUE06 GVALUEO7 Chromosome 1 Mb Figure 9 Example of gen
58. tension snpe for p values 1s required The format of the input file 1s Column 1 SNP name Column 2 chromosome number Column 3 SNP position on the chromosome Columns 4 through Column n3 Trait columns of P values for trait 1 through trait n where 0 lt n x 100 As an example the following is an input file named SNPEVGI_ inputl snpe SNPID Chr Position Tl geno Til add T1 dom T2 geno T2 add T2 dom SNPO1 1 571340 2 48E 03 Pe E DRESS 1499E 0 3 2a 125302 Z409R Ul JJ llI 03 SNPO2 1 845494 7 02E 04 9 0 7E 04 Ox 9lIE 0S Ze 00E 02 9s 02E 202 9407E 04 SNPOS 1 883895 TOTES Te TLES OA Sus D mea LE OZ Zuolm U5 du Pee Od SNPO4 Tr 929617 Le MOEDO Leo TES 02 142 9 9B 5 910 8702 22 99RAO04 eS TIED SNPO5 1 950841 L208 03 TQ T7E 03 lo32R 05 8 46E 02 Pete Vu VEU In the above file column 1 1s the marker name column 2 1s the chromosome number column 3 is the marker position in units of base pairs bp or genetic distance in centi Morgans and column 4 through column 9 are P values of genotypic effect geno additive effect add and dominance effect dom for each trait T1 or T2 in this example Any type of user defined genetic effect could be included as a trait column For example one type of genetic effect such as additive effects for all traits could be placed in one input file as shown in the following example named SNPEVGI input2 snpe SNPID Chr Position Tl add T2 add T3 add T4 add T5 add SNPO1 1 57134
59. to place all files in SNPEVGconvert zip in that folder and then change SNPEVGconvert exe file to SNPEVGconvert exe file for Windows x86 32 bit version or change SNPEVGconvert x64 exe file to SNPEVGconvert x64 exe file for Windows x64 64 bit version 2 2 Mac OS X 10 6 or newer 64 bit The installation of SNPEVGconvert only requires to unzip SNPEVGconvert Mac zip file to obtain SNPEVGconvert file 3 FILE FORMATS 3 1 Parameter file parameter dat A parameter file with the name parameter dat is required to run SNPEVGconvert The parameter file provides user specific controls and must have the name parameter dat Table 1 Example of the parameter file for SNPEVGconvert plink assoc linear input file name 5 number of columns in the output file 2 13 9 8 positions of columns to print as input file for SNPEVGI and SNPEVG2 Must be in this order SNP marker chromosome position p value 1 p value_n assoc snpe output file name WARNINGS Adding or deleting any line creates errors and is not allowed The user may change the parameter values but may not add or delete any line in the parameter file provided by the program The output file of the SNPEVGconvert program is the input file of SNPEVGI and SNPEVG2 and is the SNPE file format The output file must be in the order of SNP marker chromosome position and at least one p value which is determined in the parameter file 33 3 2 Input GWAS file The input GWAS file is d
60. ypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options Manhattan Plot Q Q Plot Line Chromosome E Trait Cut off P value 4E 7 Threshold P value Line 4E 10 Shade Points below Threshold Line Line needs to be off Unknown Chromosome 32 Sort SNP Position needs to be off Genotypic Significance Dominance Significance Additive Significance Number of Observations Lo Rn SE Save Graph s Chromosome Current Graph Current Trait Figure 5 Manhattan plot with shading of data points below threshold P value line upper and Manhattan plot with cut off P value and shading of data points below threshold P value line lower 41 Genotypic Significance Additive Significance Dominance Significance Number of Observations single locus fig Traitl ManhattanPlot I1 p1 P1 g0 mn single locus fig Trait1 Q QPlot mg single locus fig Trait1 Q QPlot ma single locus fig Trait1 Q QPlot md single locus fig Trait1 01 I1 i1 egadn p1 P1 90 s0 single locus fig Trait1 02 I1 i1 egadn p1 P1 90 s0 Input File C SNPEVG single locus fig out SNP Position C SNPEVG snp_position pos SNP Effects Genotypic Significance V Dominance Significance Additive Significance Number of Observations Advanced Options Manhattan Plot Q Q Plot Line Chromosome E Trait Cut off P value 0 0000004 Threshold P value Line 0 0000004 Shade Points below Threshold Line

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