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Integrative Genomics Viewer The Integrative Genomics Viewer is a visualization tool for exploring and analyzing large genome datasets. It is a lightweight genomic data viewer on which you can work with prebuilt genomes or load any genome that you want. It may be used for viewing a variety of data such as expression data, NGS alignments, microarray, epigenomics, RNA-Seq, genomic annotations etc. IGV has a friendly user interface. You may run it locally on your desktop or launch it from the Broad Institute website. You must register for launching or down loading. It is a Java application and you will need to install Java 6 or later on your desktop. I. Launch or download IGV 1. Go to this web page: http://www.broadinstitute.org/software/igv/download. 2. The first time you sign on, you will need to register to download IGV. 3. Click on the first launch icon (Launch with 750MB); 750MB is the smallest memory available and is sufficient for most applications. 4. Save it on the desktop as an IGV icon. When you need to start IGV, simply click on this icon. Notes: Alternatively, you may download IGV zip file; unzip it and install on your desktop. However, if you want to start IGV from command line (UNIX) you will need the binary distribution. You may also need SAM Tools and IGV Tools. These are described later 1 II. IGV User Interface A. Main Window 1 Tool Bar 2 Chr ideogram displayed in red 3 Ruler 4 Tracks 5 Genes 6 Track names 7 Attributes Access various functions Indicates the area of the chromosome currently displayed The tick marks show chromosome locations Data is displayed in tracks. Each track represents a sample or an experiment Displays features such as genes Each track is assigned a name Each attribute has a name. The values are displayed as colored blocks B. Tool Bar (Source: IGV User Guide) Genome drop-down box Loads a genome Chromosome drop-down box Zooms to a chromosome Search box Whole genome view Displays the chromosome location being shown. To scroll to a different location, enter the gene name, locus, or track name and click Go. .. Zooms to whole genome view 2 Moves backward and forward through views of the genome like the back and forward buttons in a web browser. Refresh Define a region Refreshes the display. Defines a region of interest on the chromosome Reduces the row height on all tracks to fit all data for the region in view into the window; will also expand tracks (to their maximum preferred size) to fill the view, if needed. Toggles the pop-up information windows in IGV on or off. Zoom slider III. Zooms in and out on a chromosome. Sometimes referred to as the "railroad track." IGV Exercises Following short exercises will demonstrate the various functionalities of IGV. Create a directory called igvdata and download following file for these exercises (instructions to download will be provided separately): 3 dnai1.reads.sam Ex. 1. Load a genome and define region of interest (7-9) a. Click on genome box down arrow; click on More: a number of prebuilt genomes will be displayed. b. Select A. thaliana (TAIR9) to load the genome. c. Click on Chr 1. d. Select an area on the chromosome; this will be the region of interest and will be displayed as a red block on the chromosome ideogram. e. Use the Zoom Slider (upper right) to zoom in till you see the gene sequence (bases) at the bottom of the screen. Ex. 2. Load a prebuilt genome; search for a gene, view gene sequence, translate to amino acid sequence, and copy sequence to the desktop. (10-15) f. Click on genome box down arrow; a number of prebuilt genomes will be displayed. g. Select Human HG19 to load the genome. h. Click on Chr 9. Chromosome ideogram is displayed under the Tool Bar; gene features are displayed at the bottom. i. Select an area on the chromosome; this will be the region of interest and will be displayed as a red block on the chromosome ideogram. Note the number of base pair displayed in the current window. j. Zoom in till you see the gene sequence (bases) at the bottom of the screen. 4 k. Navigate by clicking and dragging the main portion of the window in order to move left and right on the genome. l. Search for a bladder cancer gene, DAPK1. Type DAPK1 in the search box on the top and click Go. m. DAPK1 gene is displayed at the bottom. Zoom in till you see the bases. n. Right click on the gene sequence and select Translate to see the amino acid sequence. o. Right click on the chromosome and copy the sequence to the clipboard. Save it in igvdata directory. Ex. 3 Use IGV Tools to sort a file, create an index file, and create a .tdf file. i. Sort an input file – dnai1.reads.sam a. On the Menu bar click Tools > igv tools > sort. b. Click Browse and point to the input file name (dnai1.reads.sam) in the igvdata directory. c. Click run; dnai1.reads.sorted.sam will be created and saved in the igvdata directory. ii. Create an index file on dnai1.reads.sorted.sam a. On the Menu bar click Tools > igv tools > index. b. Click Browse and point to the input file name (dnai1.reads.sorted.sam) in the igvdata directory. c. Click run; dnai1.reads.sorted.sam.sai will be created and saved in the igvdata directory. iii. Create a binary tiled data file (.tdf) on dnai1.reads.sorted.sam a. On the Menu bar click Tools > igv tools > to TDF 5 b. Click Browse and point to the input file name (dnai1.reads.sorted.sam) in the igvdata directory. c. Click run; dnai1.reads.sorted.sam.tdf will be created and saved in the igvdata directory. Ex. 4 Use SAMTOOLS to convert a SAM file to a BAM file; sort the BAM file; create an index file BAM is the binary equivalent of the SAM (Sequence Alignment Map format). These formats are used for conveying alignment mapping information. BAM files are frequently used for various kinds of analyses. SAMTOOLS is an open source program, available at samtools website or sourceforge.net. It needs to be downloaded, unzipped and installed. Commands are given on the command line in the UNIX environment. If you happen to be using Windows computer, you may buy a KNOPPIX (LINUX) DVD or download Linux on a cd and make it executable. This will allow you to boot your computer from the DVD/CD and work in the UNIX environment. i. Start SAMTOOLS and make it executable with make command a. Point to SAMTOOLS-0.1.19 in the igvdata directory. b. Right click > open in terminal c. make j. Convert a SAM file to a BAM file a. ./samtools view –b –S dnai1.reads.sam > dnai1_reads.bam (Give the full path name of the input file such as: /media/sdb1/igvdata/ dnai1.reads.sam). 6 Note: You may convert SAM to BAM in Galaxy if you are familiar with it. ii. Sort a BAM file In order to create an index BAM file, it needs to be sorted first. ./samtools sort –m 1000000000 dnai1_reads.bam dnai1_reads_sorted.bam Note: Modifier –m indicates the amount of memory that can be used iii. Create an index file on dnai1_reads.sorted.bam a. ./samtools index dnai1_reads_sorted.bam b. dnai1_reads_sorted.sam.sai will be created and saved in the same directory as the input file. 7