Visualizations of Next Generation Sequencing Data in Partek
Contents
1. of all samples Profile of selected sample Bar chart of selected sample Profile of the expression of the levels of a sample attribute Category Profile Profile of the difference between two levels of a sample attribute Difference Profile C Heat map summarized by sample attribute Figure 2 Choosing tracks in the track wizard The resulting plot shows the RPKM values of all the samples four in this example as a heat map middle section while the profile of the selected sample is shown in the profile below In this example the first sample was selected and this is indicated by the sample name on the left side of the map as well as by the box around the first section of the heat map Figure 3 Also please note that the data points 1 e RPKM values in the profile track are hardly visible due to the scale of the y axis To configure the y axis scale do the following 1 1 aoo 4 92 108 55 brain 1 1 brain 3881 60 1940 80 Figure 3 Chromosome view showing RPKM values heat map represents all the samples in the original spreadsheet while the profile track represents the selected sample in this example the top sample in the heat map e Select the profile track in the list of tracks in the upper left corner The configuration options will appear in the pane in the lower left Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 2 e Set the Max y axis value according to t2. Spreadsheet and use the drop down list to specify the spreadsheet containing RPKM values Figure 1 To proceed select Next gt G W TrackWizard A i lt Select an option Add an annotation track with genomic features from a selected annotation source Add a track from spreadsheet 1 1 RNA Seq_results transcript rpkm v Select a spreadsheet then a track type The list of types depends on the content of the spreadsheet Add tracks from a list of samples Add profiles grouped by samples from every spreadsheet Add tracks from a list of spreadsheets Add one track of the default type from selected spreadsheets Add a track with the sequence of the reference genome Add a track with cytobands Other Advanced Figure 1 Selecting a spreadsheet with RPKM values in the track wizard Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 1 e Inthe next window choose the View Type Figure 2 If you want to examine samples one at a time select Profile of Selected Sample On the other hand the option Heat Map and Profile of Selected Sample allows you to visualize all the samples by a heat map and additionally to focus on the sample of your choice Depending on your preference please choose one and select Create to finish for this User Guide the latter one was chosen Choose the type of track you would like to create View Type Heat map and profile of selected sample C Heat map
3. VAResults x cancel ay Finished Figure 12 Setting up interaction between genotype calls and other factors which are hypothesized to drive gene expression Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 6 e An ANOVA spreadsheet will be created with SNVs on rows To visualize the interaction between an SNV and other factors right click on a row header and go to ANOVA Interaction Plot The resulting plot Figure 12 shows the impact of each allele x axis on gene expression y axis shows the number of genotype calls reads at the SNV position The lines represent levels of the investigated factor 1 e experimental groups In this example the G allele drives the difference in mRNA expression between the two tissue types File Edit View Window Help Ke BOM Color Auto v ANOVA 2way 4 PI gt k a chr17 7458896 A LS Mean rat 65 i l 0 5 E call a ag T Figure 13 ANOVA interaction plot showing the association of genotype between the two groups and mRNA expression at the selected locus The data points are given as the least squares LS mean with standard error End of Tutorial This is the end of the tutorial If you need additional assistance with this data set you may call our technical support staff at 1 314 878 2329 or email support partek com Last revision Sept 5 2012 Copyright 2012 by Pa
4. Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 This tutorial will illustrate how to e Visualize RPKM values for each sample in RNA Seq workflow e Visualize allele specific expression This tutorial assumes the user is familiar with the hierarchy of spreadsheets and analysis in Partek Genomics Suite PGS More details about customizing plots can be found in Chapter 6 of the Partek On line Documentation available from Help gt User s Manual from the main toolbar The data for visualizations in PGS comes from a spreadsheet If you only wish to include certain rows or columns in a plot you should apply a filter and or clone the spreadsheet or select only certain rows or columns There is no specific dataset for this tutorial you may use one of your own next generation sequencing NGS experiments or use the data from another tutorial Visualize RPKM Values in RNA Seq Workflow By default the chromosome view invoked on RNA Segq data shows raw read counts for more information please consult the RNA Seq Tutorial as well as the Chromosome View User Guide To show the RPKM values instead take the following steps e First invoke the Chromosome View showing raw reads e Delete all the Bam Profile tracks by selecting them 1n the list of tracks in the upper right corner and selecting Remove Track e Select New Track to invoke the Track Wizard In the Wizard please select the option Add a Track From
5. he maximum RPKM count also visible from the legend of the heatmap and Min to 0 e Set the Unsmoothed point size to 3 and leave the Smoothing window blank Figure 4 Select Apply to accept the changes Y Axis Color Labels Samples Min 0 Max 160 Number of grid lines 3 Smoothing window 2 Smoothed point size 3 Unsmoothed point size 3 v Draw smoothed points v Draw unsmoothed points Drop line to x axis Connect smoothed points Connect line to the start and stop of the region Line width 1 Figure 4 Configuring the profile track in the chromosome view Each dot on the plot now corresponds to the RPKM value of the respective transcript Figure 5 RefSeq Transcripts RPL22 weeeeNF207 2 m PRT Base Colors BABCHGBTaN ACOT7 erence 1 1 om 1 1 brain 20 00 10 00 0 00 Figure 5 RPKM values now visible Visualize Allele Specific Expression Allele specific expression enables the researcher to explore the association of a single nucleotide variation SNV with transcript expression Specifically are different alleles at the same locus associated with different number of sequencing reads across the groups Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 3 To start perform Detect SNVs among samples RNA Seq workflow Allele Specific Analysis gt Detect Single Nucleotide Variations The resulting spreadsheet SNVsAcrossSamples
6. rtek Incorporated All Rights Reserved Reproduction of this material without express written consent from Partek Incorporated is strictly prohibited Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 7
7. the Column Label Figure 11 Select OK to continue Visualizations of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 5 A brain C in E brain JE heart heart ao heart ijljm al r F igure 10 Result of column splitting Column Label Genotype call 4 Type categorical String Size 7 Attribute factor Random Effect Oo F igure 1 1 Changing column properties e Annotate your samples by inserting additional columns and entering appropriate labels not shown The idea of this step is to create factor columns for ANOVA e Go to Stat gt ANOVA to invoke the ANOVA dialog Enter the factors in the model and make sure to enter the factor containing the genotype calls in this example call as well as the interaction between the genotype calls and the factor whose interaction with the genotype needs to be assessed in this example tissue Figure 12 To learn more about ANOVA setup please consult our documentation Once the setup is completed select OK to proceed ANOVA of Spreadsheet 3 Experimental Factor s ANOVA Factor s 2 typ 3 tissue AddFactor gt Q Kas E ANENE 2 type 4 call lt Remove Factor 2 Save Model Load Model Contrast Cross Tabs IG Advanced Response Variable s All Response Variables v V Specify Output File _C Users User AppData Local Temp ANO
8. tions of Next Generation Sequencing Data in Partek Genomics Suite 6 6 Page 4 2 3 4 D O chr 1 18062302 chr 1 18062746 chr 1 22763642 chr Lz 2 6 6 T 323 306 323 306 0 doo 0 1 0 0 152 0 0 0 0 0 0 0 0 0 566 457 128 4 3 34 459 592 0 2 1 Jheart N 0 0 0 Alivers 98 2 0 0 diverse a 175 a a Figure 8 Result of transposition of SNVsAcrossSamples spreadsheet e Remove the row with log odds values right click the row header Delete In addition you might want to consider removing the rows showing no calls e To extract the genotype calls from the cells in the column 1 right click on the header of the column 1 and select Split Column Split the text by setting the colon as the delimiter Figure 9 Select OK to execute CP Split Text into Columns Spreadsheet 3 El Select the text column to convert 1 ID New columns are added after column 1 ID Text Split By Start from the Beginning v of the text Width Specification Example 3 5 2 Specify Width s 1 Delimiter s RE of Delimiting Times As many as possible Figure 9 Splitting a column by choosing a delimiter e Two columns will be created Figure 10 One contains the group labels while the other contains genotype calls Change the properties of both columns right click on a column header and go to Properties Set the Type to categorical and Attribute to factor In addition feel free to change
9. will have SNVs on rows and genotype calls on columns Figure 6 Please note that the SNV coordinates are given in column 1 position L De 3 t D 6 T position log odds ratio reference base brain genotype heart genotype liver genotype skeleton_muscl of different call call call A GG AA AA chr 1 18062302 323 chr 1 18062746 323 306 c GG CG oc 0 1 chr 1 22763642 323 306 A NN AG NN GG lo 0 0 chr 122763642 323 306 G NN AG NN AA lo 0 0 chr 10 1153655 323 306 c NN cc NN AA lo 0 0 chr12 6516581 323 306 T cT TT TT TT 0 570 3 chr 14 2296272 323 306 A NN m NN AG 0 o 0 F 19 4Q7R 789 374 TMA G Gea AG Gea Gea 1 1 248 Figure 6 Result of Detect SNVs across samples Each row is a single nucleotide variation SNV while genotype calls are on columns To proceed to the visualization follow the steps e Select Transform gt Create Transposed Spreadsheet and choose the SNV coordinates as column headers Figure 7 f Create Transpose Spreadsheet of Spreadsheet 2 y S Column to Get Column Labels From Choose the column whose values will be used as the column labels in the transposed spreadsheet Column 1 position Data to Transpose Data Type numeric Figure 7 Transposing the SNVsAcrossSamples spreadsheet e Observe the layout of the transposed spreadsheet an example is shown in Figure 8 SNVs are on columns log odds ratio in the 1 row while remaining rows show genotype calls per sample Visualiza
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