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integrative genome viewer manualThe menu bar and pop-up menus (not shown) provide access to all other functions. When zoomed out to display the full chromosome, the red box disappears from the ideogram. The tick marks indicate chromosome locations. The span lists the number of bases currently displayed. Typically, each track represents one sample or experiment. This example shows segmented copy number data. By default, IGV displays data in one panel and features in another, as shown here. Drag-and-drop a track name to move a track from one panel to another. Combine data and feature panels by selecting the option to display all tracks in a single panel on the General tab of the Preferences window. Legibility of the names depends on the height of the tracks; i.e., the smaller the track the less legible the name. Specify the image file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. The currently displayed reference genome cannot be removed. If no regions of interest are defined, no BED file is created. more. Users can load at.txt file that contains a list of commands, one per line, that will be run by IGV. The accepted commands are the same as the IGV Port Commands. Operators include add, subtract, multiply, and divide. For example, when multiplying two tracks, for a locus each with data values of 10 and 2 in the separate tracks will have a value of 20 in the new track. To jump to a different location, enter the locus or gene name and click Go. more. Options include displaying the information as the cursor hovers over an item, or when the item is clicked. The popup can also be disabled. Tip: Keep in mind that right-clicking an attribute may select tracks that are not visible in the data panel. Scroll down the data panel to view all the selected tracks. Most changes made via the pop-up menu are lost when you exit IGV unless you save the session.http://edouardweil.com/userfiles/impala-repair-manual.xml
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In a few cases, changing the pop-up menu also changes an option in the Preferences window; these changes are persistent. This page lists commands by track type: data track, feature track, and alignment track. Use your browser's search function to find a particular command. The following commands appear in the pop-up menu for data tracks: Select the function IGV will use to summarize the values. With autoscaling enabled, IGV automatically adjusts the plot Y scale to the data range currently in view. As the user pans and moves, this scaling continually adjusts. Restores them to separate tracks. Specify the image file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. For an example, see the Gene track, which IGV loads when you select a genome. The following commands appear in the pop-up menu for feature tracks: Change the default search server in Advanced Preferences. In other words, if you set this at 50 kb, IGV will only display features after you have zoomed in to display 50 kb or less in the IGV window. Specify the image file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. Tip: Zoom in to view alignments and the alignment track pop-up menu. This option copies that information and the read sequence to the clipboard. Low mapping quality reads are still represented in unshaded white. Translocations on the same chromosome follow the color-coding schema for pair orientation, whereas translocations between two chromosomes follow the color-coding schema for insert size. If a read pair has both unexpected orientation and insert-size, the orientation color schema is used. Displays reads or read pairs in which the forward read is first (F1 or F1R2) in red and reads or read pairs in which the reverse read is first (R1 or R1F2) in blue. Unknown status is in gray.Details are here.http://contentlock.com/personal/KarenAndSteve/chiroindex.org/web/htmls_test/userfiles/impala-owners-manual-2011.xml The rules for what constitute a mismatch to the reference genome are adjusted to account for the expected C to T (and for the reverse complement G to A) conversions. A red C or G indicates a protected site, such as by methyl modification, while a blue T or A indicates bisulfite conversion of an unprotected site. Changing this option also changes the option on the Alignments tab of the Preferences window. To change the default color scheme, see Modify the prefs.properties file. To change the default color scheme, see Modify the prefs.properties file. To generate coverage data, use igvtools. more. IGV will highlight that read with a colored border. Note that IGV does not change the view, so if the read is not currently visible this option will have no apparent effect. A black outline indicates that the selected read has no mate. Change the default search server in Advanced Preferences. The method for calculating the consensus is taken from Cavener, Nucleic Acids Res. 15, 1353-1361, 1987. Rows in the matrix correspond to the bases along the sequence. The values in a row show the counts for each type of nucleotide at that locus. A header row above the matrix indicates the order of the nucleotide columns (A, C, G, T, and N). Specify the image file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. Preferences are preserved across sessions. To override preferences during a session, use the track display pop-up menu. Each section on this page describes the options on a tab of the Preferences window: General, Tracks, Mutations, Charts, Alignments, Probes, Proxy, Advanced, and IonTorrent. Clear (default) to display both regions in the same way (white). Affects only bar charts and scatter plots. Clear (default) to display data tracks (e.g., expression data) in one panel and feature tracks (e.g., genes) in another. Clear to hide the attributes.Clear (default) to leave them without black boundaries.http://superbia.lgbt/flotaganis/1652988908 When selected (default) the zoom level is automatically adjusted so that the target feature fills the view after a successful search. If not checked, the target feature of a search is centered in the view but the zoom level is unaffected. To specify the flanking region as a percentage of feature length, enter the percentage as a negative number.IGV uses the corresponding attribute value as the track name. You may have to restart IGV for this to take effect. This option has no affect if the overlay option is not selected. To view and change the mutation coloring scheme, click the Choose Colors button.Clear to show the borders in black regardless of the track color. Tip: To change the track color, use the track display pop-up menu. As the user pans and moves, this scaling continually adjusts. Clear to turn autoscaling off. There is an option in the popup menu to enable autoscaling for a single track. Reads are visible only when IGV is zoomed in to display a number of bases less than or equal to this threshold. The coverage track, displaying total coverage at a region, is unaffected; that is, it always shows unsampled values. Default setting downsamples up to 100 per 50 nt window and paired reads are downsampled as a set. IGV uses reservoir sampling, so that all reads are kept if the read count is less than Max read count.When zoomed out, the black rectangles may appear like a continuous black line. The default is 0.2, i.e., if a nucleotide differs from the reference sequence in greater than 20 of reads, IGV colors the bar in the coverage bar chart in proportion to the read count of each base ( A, C, G, T ). The threshold for an individual track can be changed from the pop-up menu. Prior to IGV v2.3.46, allele fractions automatically displayed quality-weighted fractions. The tooltip metrics reflect nonadjusted fractions.In DNA-Seq alignments these PCR or optical duplicates are often marked and filtered. In RNA-Seq alignments considerations differ.http://futurepointtech.com/images/canon-mp145-user-manual.pdf A read may map ambiguously to multiple locations, e.g. due to repeats. Only one of the multiple read alignments is considered primary, and this decision may be arbitrary. All other alignments have the secondary alignment flag. A chimeric alignment that is represented as a set of linear alignments that do not have large overlaps typically has one linear alignment that is considered the representative alignment. Others are called supplementary and have a supplementary alignment flag. The filter file is a text file that lists read groups, one per line. This option means that IGV does not load the alignments associated with these read groups. Specify a URL or absolute file path to the file.Only alignments with mapping quality greater than or equal to this threshold are shown. A commonly used base quality metric is the Phred quality score, represented as Q, as detailed in Wikipedia. An insertion within a read is denoted with a purple I ( ) as detailed in Viewing Alignments. When this feature is activated, the I symbol is colored red for insertions larger than the size specified. At higher resolutions, the center line becomes two lines that frame the aligned bases at the center of the display, as shown in the figure above. The coverage track is visible only when alignments are visible. It displays a gray bar chart showing the depth of the reads at each locus. If a nucleotide differs from the reference sequence in greater than 20 of quality-weighted reads, IGV colors the bar in proportion to the read count of each base ( A, C, G, T ). Modifying this option affects the display of subsequently loaded alignment tracks. Note, to change the threshold from the default 20, see Coverage allele-freq threshold. This affects the coverage of displayed junctions, and the display of junctions covered only by reads with small flanking regions. Only paired end alignments with insert sizes between these thresholds are flagged. Select Compute to compute selected values from the actual size distribution of each library. If that fails, map the probe ID to a gene, determine the gene locus, and display data at that location. IGV will use this to load hosted genomes and hosted data sets. Enabling the port allows control of IGV from a web browser. more. These settings are rarely changed. On rare occasions, it may be necessary to clear the cached genome file to display an updated version of the genome. Click Clear Genome Cache to do this. Clear to disable this automatic check. This can significantly improve performance in some circumstances with running with X-Windows. Change the server hosting the genome against which BLAT searches. The default is the BLAT server hosted by UCSC's Genome Browser. Most UCSC derived genomes are supported, including human and mouse genomes. Use the Color Legends window to change the default colors for these heatmaps. An auto-complete menu will show the possible matches when a partial string is entered; if there are multiple matches IGV will show a dialog asking for clarification IGV accepts 2 mutation formats:Intially, this list contains a single item, Human hg18 or Human hg19, depending on the version of IGV.This will bring up a list of all the genomes on the server. The genomes are listed in alphabetical order. Scroll down to find the one you want, or use the 'Filter' to search. A new entry will be inserted in the drop-down list (in alphabetical order), and the display will switch to this genome. All available genomes are listed, even those that have already been loaded into the IGV drop-down menu. This is in case you want to now download the sequence for a genome already in the menu. Note that a downloadable FASTA file is not available for all hosted genomes. A notice will pop up if you try to download a sequence that is not available. An alternative is to package all the genome information into a single.genome file, as described below. IGV remembers the location of the FASTA file and the file will appear in the drop-down list until it is removed as described below. Click Save to complete. This option enables additional files to be associated with the FASTA reference sequence file, as described below. These files are archived in a zip with with a.genome extension. This option also allows the reference sequence to be defined as a directory of FASTA files, rather than a single FASTA. Prerequisites: Directories of zip archives and gzipped FASTAs are not supported. The file can be in BED format, GFF format, or any variation of the genePred table format. (Optional) Step-by-step: If the FASTA file has not already been indexed, an index will be created during the import process.Certain well-known aliases are built into IGV and do not require an alias file.This option is turned off by default but can be enabled from the Advanced tab of the Preferences window. It is good practice to read this response before sending the next command. Failure to do so can overflow the socket buffer and cause IGV to freeze. See the example below for the recommended pattern. Unloads all tracks except the default genome annotations. Specify a comma-delimited list of full paths or URLs. For example If a list is provided, these loci will be displayed in a split screen view. Use any syntax that is valid in the IGV search box. Group alignments by one of the following options: Images created from a port command or batch script are not limited to the data visible on the screen. Stated another way, images can include the entire panel not just the portion visible in the scrollable screen area. The default value for this setting is 1000, increase it to see more data, decrease it to create smaller images. The sleep interval is invoked between successive commands. If filename is omitted, writes a PNG file with a filename generated based on the locus. If filename is specified, the filename extension determines the image file format, which must be.png,.jpg, or.svg. Recognized values for the option parameter for segmented copy number are (1) for segmented copy number AMPLIFICATION and DELETION, and (2) for alignment tracks POSITION, STRAND, BASE, QUALITY, SAMPLE, READGROUP, INSERSTSIZE, FIRSTOFPAIRSTRAND, MATECHR, READORDER, and READNAME. Option values are case insensitive. If supplied, the locus option can define a single position, or a range. If absent sorting will be perfomed based on the region in view, or the center position of the region in view, depending on the option. This preference only lasts until IGV is shut down. The user loads a TXT file that contains a list of commands, one per line, that will be run by IGV. Arguments are delimited by spaces ( NOTE: not tabs ). See Controlling IGV through a Port for accepted commands. This makes use of the listener port, which must be enabled. Links can be created to load data or jump to a locus as follows. The merge parameter (optional) controls whether or not the loaded data is merged with the existing IGV session, or a loaded into a new session. If false, any data currently loaded will be unloaded after clicking this link. The default value is false if file is a session file, true otherwise. The name parameter (optional) specifies a name or names for the track. If multiple tracks are loaded as a comma-delimited list, the name parameter value should also be a comma-delimited list of the same size. The name parameter is ignored if loading a session. You can also create a session file manually. The XML format (IGV version 1.5) is described below. They are included in an XML file produce when you save a session in IGV, but are typically not included in an XML file that is created manually. Note that the sequence and the arrow are only displayed when zoomed in to a sufficiently small region. An arrow pointing left indicates that the negative strand is showing. This strand will show the complement nucleotides and reverse complement translations. The translation is shown for the strand indicated. Specify the image file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. Methionines are colored green, and all stop codons are colored red. When you zoom all the way in, the amino acid symbols will appear. These allow you to display overlapping features, such as different transcripts of a gene, on one line or multiple lines To feature-jump, you select a feature track and press Ctrl-F for forward, Ctrl-B for back. To exon-jump, you select a feature track and press SHIFT-Ctrl-F to center the next exon in your view, SHIFT-Ctrl-B to move back one exon. These attributes will be displayed in the mouse hover popup text. See File Formats for information about the file formats IGV accepts. Click the check box to the left of a dataset to select it. Warning: Selecting a folder selects all of its subfolders and all of the datasets in those folders. This can potentially be a huge amount of data. To be sure you are loading only the data you want, open collapsed folders and select only the datasets of interest. For instance, the first 2 lines of an alias file might look like this: Typically, each track represents one sample or experiment. For each track, IGV displays the track identifier, one or more attributes, and the data. Some common file formats, assumed data type, and display options are listed below. For a complete list of display options, review the options available in the pop-up menus, Preferences window, Color Legends window, and the menu bar (View and Tracks menus). IGV determines the default data range for a track as described in Default Display. The heatmap scale can be set per track. If necessary, it sets the track height to 1 pixel and scrolls the data. Legibility of the track names depends on track height; for example, track names will not be legible when track height is 1 pixel). You can preserve track name changes only by saving the session. These allow you to display overlapping features, such as different transcripts of a gene, on one line or multiple lines The commands that appear in the pop-up menu are those relevant to any data track. Data formats are described here. The Y-axis shows -log10 transformed P values, which represent the strength of association. You can see the point size difference in the following screenshot of data on chromosome 1. You can see the pop-up for the topmost data point in this image. Note that the point's position on the scale on the left is associated with its P value. The chromosome color scheme (default) uses the colors defined by IGV. Specify the file format by setting the filename extension in the file save dialog to.png,.jpeg,.jpg, or.svg. Alternative structures, where one nucleotide is involved in more than one base pair, and pseudo knots, where arcs cross, can be accommodated. Each record line must contain the first three columns of a bed file: chrom, start and end, where the start and end represent the base pair. Note that the start position follows standard BED file convention and is zero-based (first base on a sequence is position 0). The following small example represent a hypothetical stem loop: RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 2016 May 12. Overview, default coverage track, extended coverage track View as pairs, color, and split screen view Some of these preferences can be overridden on a per-track basis through pop-up menu options or by loading saved sessions. Adjust Alignment Preferences panel parameters for RNA-Seq data, PCR-free whole genome sequences, and other data that deviate from the breadth and depth of coverage of typical DNA alignments. In addition, check Show junction track to visualize splice junctions. For example, the index file for test-xyz.bam would be named test-xyz.bam.bai, or alternatively test-xyz.bai. When you specify the location of the alignment file, IGV automatically searches for the index file within the same directory. If you need to create the index yourself, there are multiple tools available for indexing BAM files, including igvtools, the samtools package, and the Picard.SortSam module in GenePattern. This setting can be altered from the Alignments tab of the Preferences window. Also, showing or hiding individual tracks can be controlled with track popup menus. This is in the General tab of the Preferences window. The Splice Junction Track is covered on a separate page. When IGV is zoomed to the alignment read visibility threshold (by default, 30 KB), the coverage track displays the depth of the reads displayed at each locus as a gray bar chart. If a nucleotide differs from the reference sequence in greater than 20 of quality weighted reads, IGV colors the bar in proportion to the read count of each base ( A, C, G, T ). For example, set the value to.3 to change the threshold to 30. Copy the count details to your computer's clipboard from the right-click menu. When this option is used the track displays coverage at all zoom levels including at the whole genome and chromosome view. To generate the extended coverage data file ending in TDF extension, use igvtools. The resulting file can be associated with the alignment track by file naming convention or loaded independently from the track popup-menu. The first occurs as the threshold zoom at which alignments become visible and the second applies to areas of deep read coverage that are downsampled. We present these two levers in this section together because the settings for each combine to impact IGV performance. Users should adjust the following default settings, tuned for DNA alignments at low coverage, for specific data types in the Alignment Preferences panel. Uncheck to turn off.The coverage track represents coverage for all the reads. For options available from the alignment track menu, including grouping, sorting and coloring options, see the alignments section of the pop-up menu page. The default visibility range threshold can be changed in the Alignment Preferences panel. At higher resolutions, the center line becomes two lines that frame the bases centered in the display, as shown in the figure above. Details are below. Genetic alternations include single nucleotide variations, structural variations, and aneuploidy. Structural variations include insertions, deletions, inversions, tandem duplications, translocations, and other more complex rearrangements. Interpretation of some of these variations are discussed briefy in this section and the next. Interpreting Color by Insert Size and Interpreting Color by Pair Orientation give more detailed explaination of read colors. IGV uses transparency to indicate quality. BED format, the. VCF format file displays structural variation. Read bases that do not match are color coded, and insertions and deletions within reads relative to the reference are marked. In addition, mismatched bases are assigned a transparency value proportional to the read quality known as the phred score. This has the effect of de-emphasizing low quality reads. This is a new feature starting with IGV v2.3.46, released March 2015. Interpretation of this mapping quality depends on the mapping aligner as some commonly used aligners use this convention to mark a read with multiple alignments. In such a case, the read also maps to another location with equally good placement. It is also possible the read could not be uniquely placed but the other placements do not necessarily give equally good quality hits. Hover over the insertion symbol to view the inserted bases. For a description of all user-specified color and sort options, see the alignment track pop-up menu. IGV sorts the alignments that intersect the center line of the display. This can cause the alignment layout away from the center line to appear sparse. To restore the layout to an optimally packed configuration, select Re-pack alignments from the pop-up menu. This section covers viewing reads as pairs, coloring of mapped paired reads, and the split-screen view. Interpretation of colors is discussed briefy here and in more detail in Interpreting Color by Insert Size and Interpreting Color by Pair Orientation. Select View as pairs from the right-click menu to display pairs together with a line joining the ends as shown in the image below. The hover element details (2) are also displayed either for a single read in normal view (left) or for a pair of reads in paired reads view (right). A black outline indicates that the selected read has no mate. Turn on the Color by insert size and pair orientation option from the popup menu to confirm as described below. However, outlining reads only works in the unpaired view and not in the paired view. Note that coloring by insert size is a feature designed originally for DNA alignments against the genome. It is based on set base pair values or computed from the size distribution of a library. Right-click over an alignment and select View mate region in split screen from the drop-down list. If the alignment clicked over does not have a mapped mate this option will be grayed out. You can select this option for mutliple alignments and view multiple panels side by side. Alternatively, double-click the locus name. Pan by click-dragging in the panel; double-click to zoom in and alt-click to zoom out. It is based on set base pair values or computed from the size distribution of a library against the reference genome as defined in the Alignment Preferences Panel. This is due to the deleted section of the genome, not present in the subject. Schematically this can be visualized as follows: In IGV such an event might look like the following. They must be long enough to span the insertion and include sequences on both ends that are mapped to the reference. The maximum detectable size is approximately equal to: For instance, in this case, one end is on chromosome 1 and the other is on chromosome 6. The six offered modes are summarized in the table, and are explained further on this page. The mode supports visualization of alignments from the following and similar techniques: Pervasive in human embryonic stem cells (Lister 2009). For example, in H1 stem cells comprises almost 25 of all cytosines at which DNA methylation is identified compared to 99.98 mCG in IMR90 cells. For paired reads, this is the same as coloring by first-of-pair strand.When comparing two samples, a change in methylation status will be marked by a difference in color for a given site. Red to blue indicates loss of methylation, or hypomethylation; blue to red indicates increased protection by methylation, or hypermethylation, as shown for the tumor sample in the screenshot below which visualizes data from Berman et al (2012). Alignments in IGV are against a reference genome of correct sequence as coloring is based on deviations from the reference sequence. Read alignment may have been against a bisulfite-transformed genome sequence, in which case genomic coordinates would still be for that of the original reference genome. In DNA methylation, the methyl CH 3 group is added to the cytosine base at the carbon 5 position (5-meC) in a sequence-context dependent manner. In mammals this context is typically CpG dinucleotides, and in plants this is CpG, CpHpG, and CpHpH di- and tri-nucleotides. These correspond to the CG, CHG, and CHH bisulfite coloring modes in IGV. The IUPAC ambiguity code H represents any nucleotide but guanine. Cytosine undergoes conversion to uracil whereas 5-meC is unmodified and remains intact. The uracil is subsequently converted to thymine after PCR amplification while 5-meC residues remain cytosines. These include whole-genome bisulfite sequencing (WGBS) and reduced-representation-bisulfite sequencing (RRBS), both of which provide single-nucleotide resolution. However, as with any technique limitations exist, including the inability to discriminate 5-meC from 5-hydroxymethylcytosine (5-hmeC) modifications, which was discovered to be pervasive in mammalian DNA in 2009 (Yu, Cell 2012). Of relevance to coloring by bisulfite mode in IGV is TAB-Seq (Tet-assisted bisulfite sequencing), in which 5-hmeC sites are protected by glucosylation prior to bisulfite conversion. Because 5-meC sites remain unprotected from mTet1 oxidation to 5-carboxylcytosine (5-caC), and subsequent bisulfite conversion, only 5-hmeC site cytosines remain unchanged in reads (Yu, Nature Protocols 2012). These are the original top strand (OT), the original bottom strand (OB), and strands which are complementary to OT and OB (CTOT and CTOB). IGV visualizes reads in one direction, and for the given direction reads from the opposite strand are automatically displayed as the reverse complement. Therefore, OT and CTOT reads are displayed in the reference-forward direction (gray) while OB and CTOB reads are displayed in the reverse direction (sage) and are differentially colored as indicated. OT and CTOT yield methylation information for cytosines on the top strand ( C and T highlighted), while OB and CTOB will give methylation information for the paired complement, that is for guanines paired to the methylatable cytosines ( G and A highlighted). For example to determine nucleosome positioning in yeast and mammalian cells. This method obtains nucleosome positioning information based on the GpC methyltransferase M.CviPI accessibility to GpC sites, and at the same time obtains endogenous DNA methylation information from CpG sites. GCG is excluded due to ambiguity between endogenous and enzymatic methylation.