Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6

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1 Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 Researchers often have lists of genes, probes, transcripts, SNPs, and genomic regions from other analysis tools, research papers, or a hypothetical list of interest and wonder what they can do with such lists in Partek Genomics Suite (PGS). This tutorial will illustrate how to: Import various kinds of lists and associate properties with the list Identify which functions in PGS might be useful for that kind of list Use imported lists as filters for other PGS spreadsheets Integrate multiple types of list data The user is expected to be familiar with the PGS workflows for data analysis of typical data, that is importing microarray data from a supported chip and/or next-generation sequencing data import from SAM/BAM files. This tutorial will not explicitly discuss statistical analysis of imported data and discusses some operations that be performed on these lists. Importing a text (tab-separated or comma separated) list The best way to import a generic list of data into PGS is to use the File > Import > Text (.csv.txt) feature. If your data is stored in Excel, you should save it first as a text (tab-separated) or comma-separated file. As shown in Figure 1, the columns in the input file can be separated by a tab, comma, or by any other character. Figure 1: Choosing the type of your "list of interest" to import The imported list is easier to work with if the items in the list are on rows. If the list is oriented with the data of interest on columns, you may check the box next to Transpose the file to. If there are missing numerical values or empty cells in your input list, insert a special character or symbol (?, N/A, NA, etc.) in the missing cells; you will specify the character in the Missing Data Representation section of the dialog shown in Figure 2. The next step is to identify where the data starts and where the optional header is found. As shown in Figure 2, the line that contains the header (if present) must precede the data. If there are lines to be skipped in the file (like comments), they may only appear at the top of the file, before the header line or data begin. If there are many comment lines at the start of the file, you may need to select View Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 1

2 Next 5 Records to get to the row that contains the column header. If you accidentally move past the screen that contains the header or data rows, then select View Previous 5 Records. Figure 2: Identifying the header line and where the data starts If a header line is present, make sure the tick box next to Col Lbls is checked as shown in Figure 2. Next identify the row that contains the header line by selecting the left-most radio button next to the row that contains the header. If no header line is present, deselect the tick box next to Col Lbls. The radio button in the Col Lbls column will still be selected but can be ignored Select the radio button Begin Data at the line where the data begins. All of the lines after the line indicated by the Begin Data position will be considered data lines. No comment lines may follow the data If some of the lines contain missing data (empty cells), then you can signify the value is missing with a special symbol in the Missing Data Representation panel. This is important if the missing value is a number in a column on which you would like to perform calculations. The default missing value indicator is a? Select Next to continue with the import process The next dialog box (Figure 3) is probably one of the most important steps during the import process because it is easier to specify the variable types correctly now than to change the variables individually later. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 2

3 Figure 3: Specifying the types of data. Multiple columns may be selected during this dialog; later the properties of a column must be edited individually. If there is an identifier like gene symbol or SNP, the Type field for that column should be text and Attribute should be label. Numeric values (intensities, p-values, fold-changes, etc.) should have Type set to double and Attribute set to response as shown in Figure 3. The other possible value for Attribute is factor and is used in PGS to describe sample data, so it would rarely be used in this kind of import. The user interface is this dialog allows you to select multiple columns at once (see the outlined box in Figure 3). Starting with a List of Genes There are many useful visualizations, annotations, and biological interpretations that can operate on a gene list. In order for these features operate on an imported list, an annotation file must first be associated with the gene-list. Additionally, many operations that work with a list of significant genes (like GO- or Pathway-Enrichment) require comparison against a background of non-significant genes. The quickest way to accomplish both is to use the background of all genes for that organism provided by an annotation source like RefSeq, Ensembl, etc. in pannot (Partek annotation), gff, gtf, bed, tab- or comma-separated format. If the file is not already in a tab-separated or comma separated format, you may import, modify, and save the file in the proper file format. Select File > Import > Genomic Database (Figure 4) and choose an annotation file that contains the annotations of interest. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 3

4 Figure 4: Importing a species-specific database containing "all" genes and transcript For instance, the Partek-supplied hg19_refseq_v2.pannot was selected from the C:\Microarray Libraries folder and resulted in the spreadsheet shown in Figure 5 Delete or rearrange the columns as necessary. For instance, put the column that corresponds to the gene list first Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 4

5 Figure 5: RefSeq hg19 imported as a Genomic Database rearranged so Gene is in the first column Save the modified annotation file as a text file by clicking on the annotation spreadsheet in the spreadsheet navigator and selecting File > Save As Text File Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 5

6 Close the annotation spreadsheet by selecting Right-click on the imported gene list and select Properties. This brings up the dialog box shown in Figure 6 Figure 6: Configuring the properties of an imported gene list Several different properties will be specified including species, Gene Symbol Column, and genomic annotations. Select Other Select species from the Add Property menu and then Add In the Edit Genome dialog, specify Species Name, Genome Version, UCSC Species Name, Cytoband file, and 2bit sequence file and select OK Next, select Genomic from the Add Property pull-down and Add This will bring up the Configure Genomic Properties dialog shown in Figure 7. Select Browse and choose the text annotation file created earlier. This will allow you to specify the chromosome number, start, and stop locations according to how the annotation file was created Figure 8 Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 6

7 Figure 7: Specifying the text annotation file Figure 8: Choose the radio button that matches the format of your annotation file Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 7

8 In this case, Marker ID should be the same as the name of the gene. Select the radio button that describes how the chromosome, start, and stop positions were specified in the annotation file. Also be sure to select the radio buttons above the appropriate columns. Select Close Select Set Name Column to specify the proper annotation for the Gene Symbol column and select OK Make sure Species is still set appropriately then select OK (several times) Save the imported gene list spreadsheet (File > Save or ) Annotating imported Gene Lists Inserting Annotation Columns If the genomic annotation file included other annotations in addition to the genomic coordinates (Transcript IDs, species, etc.), the annotations may be inserted into the imported spreadsheet simply by right-clicking on a column header and choosing Insert Annotation. Any of the annotation columns may be selected. Annotate with Cytobands Tools > Annotate with Cytobands will add a column to the imported gene list spreadsheet that contains cytoband locations if both genomic coordinates and a cytoband file were associated when the gene list was imported. Annotate with Known SNPs Tools > Annotate with Known SNPs will add a column of SNPs associated with a particular gene and a column indicating the number of SNPs known to be associated with the gene. To generate a list of SNP ids per row, right-click on a row header and select Create list of dbsnp. However, this feature can associate ANY data with list of genes or genomic coordinates: the choices include the dbsnp database, any mirna database, data from the Database of Genomic Variants (dgv), any mrna transcriptome database, or any custom annotation source. This feature will add columns to the imported gene list spreadsheet that correlate the genes with the features from those databases. GO Enrichment The GO Enrichment p-value calculation uses either a Chi-Square or Fisher s Exact test to take into account which genes are in the significant gene list against which genes are in the background. For a microarray experiment, the background consists of all the genes on the chip/array; for a next generation sequencing experiment, all of the genes in the transcriptome are considered background. Figure 9: Gene Ontology Enrichment calculates (for each GO term) the statistical probability of the number of genes in the input list sharing that GO term appearing against the background of genes in the experiment Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 8

9 Because the calculation is essentially measuring overlaps of genes and does not use intensity values, Gene Ontology (GO) Enrichment can be performed on an imported gene list provided that an appropriate list of background genes is specified in the annotation step described in Starting with a List of Genes. You will need to set the Workflow to Gene Expression to access GO Enrichment. Note: The choice of background genes will affect the statistical significance of the enrichment scores. For ease of use, this User Guide has suggested using annotations from a full transcriptome which is done for next-generation sequencing experiments. However, if the imported list of genes originated from a microarray experiment, it might be better to use the annotations for that microarray for the background genes. Please contact our technical support department for assistance with this step if help is needed. Pathway Enrichment Like GO Enrichment, Pathway Enrichment does not require numerical values but operates on lists of genes: significant genes vs. not significant or background genes. Consequently, Pathway Enrichment may be used with an imported list of genes and if the imported list of genes has been associated with a list of background genes as outlined in the Starting with a List of Genes. Starting with a Gene List including Numeric Data Import and annotate the list as described in Starting with a List of Genes. All of the same operations described for gene lists are applicable; you may also use the numeric data associated with the genes for visualization, clustering, or statistical operations. Descriptive Statistics If you have imported numerical data with your gene list, you may perform statistics on the imported data. Examine the options available from Stat > Descriptive or Stat > Correlate. Principal Component Analysis may be performed from the Tools > Discover menu. Applying Multiple Test Correction If the imported data contains a list of p-values, you may use the Stat > Multiple Test > Multiple Test Corrections to apply any of the available multiple test corrections to the list of p-values. Plotting numeric data associated with a gene list To see a profile plot of numerical data associated with your imported gene list, you may use View > Profiles > Row/Column Profiles or any of the other View options. Genome Browser If you have imported numerical data associated with genes (like p-values or fold-change), you may visualize these values in the Genome Browser once the annotations have been specified. From the imported gene-list spreadsheet, right-click on a row header and select Browse to Row If the annotations have been configured properly, you should see a track for the first column of numerical data, a cytoband track, an annotation track (RefSeq in this case) as shown in Figure 10 You may add another track to display a second column of numerical data by selecting New Track > Add a track from spreadsheet (the imported gene list), and Next. Then select the track in the track navigator and select the other column in Bar height by Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version 6.6 9

10 Clustering Figure 10: Chromosome view showing numeric data associated with gene list If it makes sense to cluster the data associated with the gene list, you should use the Tools > Discover > Hierarchical Clustering command rather than Cluster based on significant genes from the workflow. The hierarchical clustering command from the workflow assumes that the data to be clustered (usually intensity values) are found on a parent spreadsheet and the list of genes is in a child spreadsheet. Since the imported data to be clustered is all in one spreadsheet, use the Tools > Discover > Hierarchical Clustering feature instead. Consider transposing the spreadsheet if samples are on columns and genes are on rows. If you only have one column or one row of data, then cluster only on the dimension with multiple entries by deselecting either Rows or Columns from What to Cluster or consider using View > Intensity Plot. Starting with a list of SNPs A list of SNPs using dbsnp IDs can be imported as a text file and associated with an annotation file just as the genes are imported and associated with an annotation source in Starting with a List of Genes. The annotation file (genomic database) you use to annotate the SNPs should minimally contain the genomic coordinates (chromosome number and physical position) of the locus. Please be aware that using hg19_dbsnp_v2.pannot as the annotation source is very memory intensive due to the file s large size. Including the genomic coordinates of the SNPs if you have them (see Starting with a list of Genomic Regions) saves having to look-up genomic coordinates from the dbsnp name. Novel SNPs, or SNPs that are not found in your annotation source, must be imported as a region list. The only difference would be to use the SNP name in place of a region name. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

11 Annotating SNPs with Genes Starting with a list of SNPs that has been associated with genomic coordinates, you may use the Tools > Find Overlapping Genes feature to annotate these SNPs as shown in Figure 11. Figure 11: Imported SNP list annotated by Find Overlapping Genes. Columns 1-9 were inserted by Find Overlapping Genes Once you have generated a list of genes, add the Gene Symbol Column property to this spreadsheet (right-click the spreadsheet > Properties > Add Property > Gene Symbol Column). Now all of the operations that can be performed on gene lists will work on the annotated SNP list. Annotating Partek-generated SNP list with SNVs If you have a SNP spreadsheet that was generated by Partek, you may annotate the SNP list with gene, transcript, exon, and information about the effect of the SNPs (Figure 12). With the SNP list selected in the spreadsheet navigator, select Tools > Annotate SNVs. Figure 12: SNP list annotated with transcript, genes, exons, and implications of the SNP Starting with a list of Genomic Regions Suppose you have a list of genomic regions that correspond to a list of copy number regions, methylation peaks, or transcription factor binding peaks. Or perhaps you have multiple lists of Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

12 genomic regions from several experiments and would like to compare the lists to determine which regions are in common. There are many features in PGS that can be used on such lists. Importing Region Lists A region list in PGS must contain the chromosome, start location, and stop locations as the first three columns, respectively, in the file as shown in Figure 13. The chromosome name (or number) in the region list must be compatible with the genomic annotation for the species if you plan to use any feature (like motif detection) that requires reference sequence information. Figure 13: Minimum amount of data required to import as a region list. The first three columns must contain chromosome name/number, the start location, and the stop location. Additional columns may follow and should be coded as text or float (if a decimal number) or integer Import the region list as described in Importing a text (tab-separated or comma-separated) list. The chromosome number or name should be imported as a text field; location start and stop may be either integer or text Right-click on the spreadsheet in the spreadsheet navigator and select Properties If you see a see a dialog box that looks like Figure 14, select List of genomic regions (eg:.bed file) Figure 14: Adding a property to an imported region list Next select region in the Add Property pull-down menu and select Add. The spreadsheet property will now include region as shown in Figure 15 Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

13 Figure 15: Properties after adding region to a newly imported region list If you plan to do any operation that requires looking up the reference genomic sequence information for the regions based on genomic location, you will need to specify the species for this region list. Select Genomic from the Add Property pull-down menu and select Add. Under Species, either choose the species from the pull-down menu or type in the name of the species if it does not appear in the list. If you wish to use the Genome Browser, then select Edit Genome to specify the Genome Version, USCS Species Name, Cytoband file, and 2Bit sequence file. Select OK Motif Detection Starting with a region list, you may detect either known or de novo motifs using the ChIP-Seq workflow if your spreadsheet has been associated with a species and a reference genome as described in the import section. Set Workflows to ChIP-Seq In the Peak Analysis section of the workflow, select Motif detection. Both options (Discover de novo motifs and Search for known motifs) can now be performed Motifs (de novo) may be displayed in the Genome Browser and known detected motifs may be viewed in web-log format by right-clicking on a header row of the motif spreadsheet. Determining the Average Values for a Region List If you have a region list or a bed file and you have a microarray experiment with data, you can summarize the data according to the genomic coordinates contained in the region list. For instance, the region list contains a list of CpG islands, the experiment contains methylation percentage values for probes ( values), and you would like to summarize the methylation values for individual probes for the CpG islands. Or you have a list of copy number amplifications, microarray gene expression data, and you are interested in determining if the average intensities of the probes in those regions is higher than expected. Import the region list (or BED file) and specify the region property as explained elsewhere in this document With the region list spreadsheet selected, right-click in any column header and select Insert Average A dialog box similar to that shown in Figure 16. With the Add Average tab selected, specify the location where you like the new columns to appear by using the Add to the and of Column pull-down menus. Specify the top-level spreadsheet containing the data you wish to be averaged ( values, gene-intensity values, etc.) in the Get average from spreadsheet pull-down menu. Choose the radio button to specify how the averaging should be done. The bottom two choices (Mean of all samples and Mean value for all samples separately) are obvious; the first option (Mean of samples significant in region) is used when the region list has a SampleID associated with each region. In this case, the column designated as the SampleID column from the top-level spreadsheet will be used to identify the sample to be summarized for each region. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

14 Figure 16: Inserting the average values from another spreadsheet based on a region list Find Region Overlaps You have a list of regions from another analysis program (perhaps you detected peaks using an R program) and you d like to compare that region list with a region list that Genomics Suite calculated. Perhaps you have two lists created by Genomics Suite (one generated from peak detection with one set of parameters and the other created with different parameters) and you d like to see what the two lists have in common. You may use the Tools > Find Region Overlaps command to compare two or more region lists as shown in Figure 17. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

15 Figure 17: Tools > Find Region Overlaps dialog There are two separate modes of operation for this command: Report all regions and Only report regions present in all list. The first option, Report all regions, will report all regions in both lists. If there is any region overlap between the lists, the intersection of the regions will be reported along with the start and stop coordinates of the intersection, the percent overlap between the intersected region with each of the regions in the input lists (left panel of Figure 18). If a region is found in only one list, it will be reported as well (right panel of Figure 18). Figure 18: Report all regions will report all regions in both lists and the percent overlap for each region list. In the panel on the left, the red region in ListA partially overlaps the orange region in ListB. The resulting overlap is shown in gray and reflects the intersection of both regions (ListA overlap=86.55%; ListB overlap=39.23%). In the panel on the right, there is no region in ListA in this area so the gray region is the exact size of the orange region in ListB; the overlap percentage for ListA=0% and for ListB=100% In contrast, the second option, Only report regions present in all lists, will intersect both lists and only reports regions found in all the lists (Figure 19) Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

16 Figure 19: Only report regions found in all lists will only report regions in common to both lists and will subdivide the region if needed. In the panel shown on the left, the red region belongs to ListA, the larger orange list belongs to ListB, and the gray region is the reported intersection of these two regions. In the panel on the right, there is only a region in ListB so no regions is shown in the gray track Importing Genomic Locations to be used with Annotating SNVs The Tools > Annotate SNVs feature requires four columns of data per genomic location: the position of the SNP (chr.baseposition), the SampleName, a reference base, and the SNP call (single nucleotide or genotype) as shown in Figure 20. Figure 20: Input file for annotating SNVs Prepare input list as shown in Figure 20 and save as either a tab-separated or commaseparated file Use File > Import > Text to import the table. During import, change the data type of column 1 (as in Figure 3) to text by right-clicking on the color bar of column one and changing the data type to text. You may leave the other columns as categorical response types The correct properties must be set for this spreadsheet. Right-click on the newly imported spreadsheet in the navigator and select Properties Choose Other in the Configure Spreadsheet dialog (Figure 6) Make sure Genomic is selected in the Add Property pull-down menu and select Add Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

17 In the next dialog box, select Genomic location instead of marker IDs in the Choose the type of genomic data. The Marker ID in column should be set to the first column. [If Marker ID in column does not contain any items in the pull-down list, it is likely that the first column was not a text column (drawn in gray) during import. If this happens, then right-click in the column header in the spreadsheet and change Type: to text.] Specify the Species from a pull-down menu selection or by typing in the species name. Select Edit Genome to specify the Species Name, Genome Version, Cytoband file, and 2Bit sequence file. The last two fields are optional. Select OK Now that the properties have been set appropriately, Tools > Annotate SNVs may be invoked on this spreadsheet. Importing a BED File A BED (Browser Extensible Data) file is a special case of a region list: it is a tab-delimited text file and the first three columns of BED files contain the chromosome, start, and stop locations. To import a bed file to be used as a data region list, follow the import instructions for region lists. A BED File might also be visualized as an annotation file containing regions in the Genome Browser. Using a BED file as an Annotation Source for the Genome Browser BED files do not contain individual sequences nor do the regions have names. For instance, the UCSC table browser has a BED file that contains reads from a long non-coding RNA-Seq experiment and you might like to view this information in the context of your dataset. Before you could visualize a BED file in the chromosome viewer, you would have to create a Partek annotation file from the BED file. From the top command menu, select Tools > Annotation Manager In the My Annotations tab, select Create Annotation Select BED file (.bed) under Choose Annotation Type Under File Locations, specify the Source (input BED file) with the Browse menu item. You may also specify the Result file name (of the annotation file) and location with the Browse button. You might consider saving the result file to your Microarray Libraries folder In the Annotation Details section of the dialog box, specify the Name of the annotation database that will be visible from within Genomics Suite, Species, and Genome Build. Preview Chromosome Names would be used if the chromosome names in the annotation file must be changed to match the name of the chromosome in the genomic annotations. Select OK Visualizing a BED file as an Annotation Track in the Genome Browser In order to use a BED file as an Annotation track in the Genome Browser, first create the annotation file as described above, being careful to have specified the species and genome build appropriately. Invoke the Genome Browser by right-clicking in any spreadsheet that has genomic features on rows (gene lists, ANOVA results, SNP detection) and select either Browse to Row or Browse to Location In the Track Toolbar on the left, select New Track which invokes the dialog shown in Figure 21 Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

18 Figure 21: Choosing which kind of track to insert in the Genome Browser Select Add an annotation track with genomic features from a selected annotation source and select Next Next you will have to choose the annotation file that was created from the BED file. The procedure for doing this might vary slightly depending on the type of spreadsheet you have displayed in the Genome Browser. You may be shown a list of annotations that includes the annotation source you have created; in this case, select the radio button in the Available Annotations panel. If, however, At the bottom of the screen, you should either check or uncheck the box next to Separate strands (checking means that the BED file contained the strand information for each region and you wish to visualize the regions on different strands). Unselecting Separate strands should be used if the BED file did not contain strand information or if you do not wish to display the information on separate strands. Select Create Figure 22: Annotation from BED file is shown in the bottom track. Notice that there are several long non-coding RNA reads in the area between the FRAS1 and ANXA3 genes. Since the BED file only contains regions, mousing over the regions will not provide any additional information Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version

19 GO ANOVA, GSEA/GeneSet ANOVA, and Pathway ANOVA As these features require intensity (or count) data as well as experimental groups, these features cannot be performed on an imported lists. Integrating Imported Data If the data from imported spreadsheets has been associated with annotations, several integration approaches may be used to integrate multiple kinds of imported data. For instance, the Genome Browser may be used to display data from multiple spreadsheets/experiments regardless of the type of spreadsheets (imported data or microarray or NGS experiments). The Venn Diagram tool may be used to find overlaps based on a feature name. Tools > Find Overlapping Regions can use an imported gene list and a list of regions from a copy number or ChIP-Seq experiment to identify genomic regions in common. End of User Guide This User Guide did not discuss every operation that can be performed on an imported list of regions, SNPs, or genes. If there is some other feature in PGS that you would like to apply to an imported list, please contact the technical support team for additional guidance. If you have found a novel use of PGS feature on an imported list that you think should be included in this User Guide, please let us know. If you need additional assistance with these steps, you may call our technical support staff at x350 or us at: support@partek.com. Last revision: Jul. 3, 2012 Copyright 2012 by Partek Incorporated. All Rights Reserved. Reproduction of this material without express written consent from Partek Incorporated is strictly prohibited. Tips & Tricks: What you can do with an imported list in Partek Genomics Suite version