Viewing Alignments

This page introduces viewing alignment data and associated tracks in the following sections:

• File Formats
• Read Coverage Track
• Downsampling
• Alignment Track
• Paired-end Alignments

Related topics on other pages cover more detailed topics:

• Alignment Preferences panel settings
• Alignment track pop-up menu options
• Interpreting Color by Insert Size
• Interpreting Color by Pair Orientation
• Interpreting Color by Bisulfite Mode
• Splice Junctions Track
• Sashimi Plots

File Formats

Aligned reads from sequencing can be loaded into IGV in the BAM format, SAM format, or CRAM format.

Both BAM and SAM files are described on the Samtools project page http://www.htslib.org and in the 2014 article titled Sequence Alignment/Map Format Specification by the SAM/BAM Format Specification Working Group.

IGV requires that BAM and CRAM files have an associated index file.

• The main data file must include the .bam or .cram extension.
• The index file should have the same filename but with the .bai or .crai extension. For example, the index file for test-xyz.bam would be named test-xyz.bam.bai, or alternatively test-xyz.bai.
• When loading by URL, the URL to both the data file and the index file should be specified.
• When loading by file, IGV automatically searches for the index file within the same directory as the data file.

If you receive a .bam file from a sequencing facility, you will usually also get the corresponding index file. 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.  

Tracks

• Alignment Track to view individual aligned reads
• Coverage Track to view depth of coverage
• Splice Junction Track which provides an alternative view of reads spanning splice junctions

Coverage Track

By default IGV dynamically calculates and displays the default coverage track for an alignment file. 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).

Pre-computed Coverage Data

The dynamically calculated coverage data can be augmented by loading pre-computed  coverage data from a file.  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. 

• To associate a coverage track using filename, the track must be named <alignment file name.extension>.tdf, and placed in the same directory as the alignment track.
  • For example, the coverage track for test.bam would be named test.bam.tdf. IGV loads this coverage track automatically when test.bam is loaded.

• To dynamically associate coverage data with a BAM track, right-click on the coverage track and choose Load pre-computed coverage data from the pop-up menu. 

Visibility Range Threshold and Downsampling

IGV reduces memory usage in the following two ways to improve performance of viewing alignments.

• A visibility range threshold defines the size of the region in view at which alignments are loaded. If the region is view is larger than this threshold, no alignments are visible. Default: 30 kb
   
• In areas of deep read coverage, by default the reads are downsampled, i.e. not all the reads are displayed. The level of downsampling is controlled with the parameters:
  • Max read count; default 100
  • Per window size; default 50 bases

You can adjust the above settings in the Alignment Preferences panel. For example, for lower coverage data, you can provide a larger visibility range threshold. Or for deep coverage, you might want to provide a smaller visibility range threshold and adjust the downsampling to show more reads.

Downsampled reads areas are marked with a black rectangle just under the coverage track. The coverage track represents coverage for all the reads.

In the example shown, the downsampled regions are marked by seven black rectangles just under the coverage track.

Alignment Track

This section gives an overview of the alignment track. For options available from the alignment track menu, including grouping, sorting and coloring options, see the alignments section of the pop-up menu page.

• When zoomed in to the alignment read visibility threshold, by default 30 KB, IGV shows the reads. The default visibility range threshold can be changed in the Alignment Preferences panel.

• When zoomed in sufficiently, IGV can display a line at the center of the display. At higher resolutions, the center line becomes two lines that frame the bases centered in the display, as shown in the figure above. 
  • The framed bases are the basis for Sort by operations for alignment tracks (see details below).
  • Enable the display of the center line by setting the Show center line property in the Alignment Preferences panel.

Detecting Structural Variants

IGV uses color and other visual markers to highlight potential genetic alterations in reads against a reference sequence. 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.

An additional factor to take into consideration when judging potential genetic alterations is quality of reads and quality of mapping. IGV uses transparency to indicate quality.

• For RNA-Seq, TopHat outputs separate insertions.bed and deletions.bed files which IGV will load as separate tracks.
• In addition to the .BED format, the .VCF format file displays structural variation.

Colors and transparency are used at two levels within alignments: (1) for mapped reads, and (2) for individual bases within reads.

Color and Transparency for Individual bases

By default, read bases that match the reference are displayed in gray. Read bases that do not match are color coded, and insertions and deletions within reads relative to the reference are marked. Insertions are indicated by a purple I () and deletions are indicated with a black dash (–). 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.

• To color code all bases, regardless of whether they are mismatched, right-click the track and select Show All Bases from the pop-up menu.  
• To mark insertions greater than a specified size with a red I, select and specify the size cutoff for the Flag insertions larger than parameter in the Alignment Preferences panel. This is a new feature starting with IGV v2.3.46, released March 2015.
• Transparency shading of quality can be turned off temporarily from the pop-up menu, or persistently from Alignment Preferences panel.
• To change the default nucleotide coloring scheme for reads, see Modify the prefs.properties file.

Transparency for Mapped Reads

Note that alignments that are displayed with light gray borders and transparent or white fill, as shown in the screenshot, have a mapping quality equal to zero. 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. 

Insertions

In a gapped alignment, IGV indicates insertions with respect to the reference with a purple I () or red I for  insertions greater than a user activated and specified cutoff.  Hover over the insertion symbol to view the inserted bases.

Deletions

In a gapped read, IGV indicates deletions with respect to the reference with a black bar.

Coloring and Sorting Alignments

Users can also specifiy color and also sort reads by various options, including start location, strand, nucleotide, mapping quality, sample tag, or read group tag. For a description of all user-specified color and sort options, see the alignment track pop-up menu.

For example, to sort alignments:

1. Right-click a track for the pop-up menu.
2. Select a Sort option from the pop-up menu. IGV sorts the alignments that intersect the locus at the center of the track, no matter where the cursor was located for the right-click. To display a line down the center of the track, set the Show center line property in the Alignment Preferences panel.

Sorting rearranges rows so that alignments that intersect the center of the display appear in the order specified.  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.

Repeat the most recent sort with hotkey ctrl-s.

Paired-End Alignments

IGV provides several features for working with paired-end alignments. 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.

View As Pairs

By default, IGV displays reads individually because they pack compactly. 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).

Coloring of Mapped Paired Reads

IGV colors paired-end alignments in two ways.

• Interactively by user selection as shown in the purple highlighted reads marked by (1) in the images above, and
• That deviate from expectations as marked by (3) in the image above.

	Control+click (Mac: Command+click) a read to outline the read and its paired mate in the same color. Colors are arbitrary but unique to each pair. A black outline indicates that the selected read has no mate. Control+click (Command+click) either read to clear the outline. Right-click and select Go to Mate Region to jump to the paired mate. If the paired reads have a large insert size, the paired mate will not be highlighted. Turn on the Color by insert size and pair orientation option from the popup menu to confirm as described below. Right-click and select Clear Selections to clear all outlines. Outlined paired reads are preserved when switched to View as pairs option. However, outlining reads only works in the unpaired view and not in the paired view.
	Hover over or click a read to view information about the read, including the location of its paired mate.
	IGV colors (1) paired end reads with inferred insert size smaller or larger than expected; (2) read with mate that is aligned to a different chromosome; (3) paired-end alignments with deviant pair orientation. 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. See Interpreting Color by Insert Size for more detail. Blue is for inserts that are smaller than expected Red is for inserts that are larger than expected. Inter-chromosomal rearrangements are color-coded by chromosome. See Interpreting Color by Pair Orientation for more detail. Shades of green, teal, and dark blue show structural events of inversions, duplications, and translocations. Color assignments depend on sequencing platform. Other Color by options are described in the alignment track pop-up menu options Translocations on the same chromosome can be detected by color-coding for pair orientation, whereas translocations between two chromosomes can be detected by coloring by insert size. See both by selecting the Color alignments by> insert size and pair orientation option.

Split Screen View

Split screen views can be invoked on-the-fly from paired-end alignment tracks. 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.