Viewing Alignments

This page outlines viewing alignment data and its components on IGV and includes directions on creating an extended coverage track in TDF format.

Additional details on interpreting colors, splice junctions, and sashimi plots are given on separate pages linked as daughter pages to this page on the left menu and at the very bottom of this page.

The following links give details on alignment pop-up menu options and the alignment Preferences window.

File Formats

The preferred file format for viewing alignments in IGV is the BAM format, a binary form of Sequence Alignment Map (SAM) format. We especially recommend the BAM format for large alignment files.

GOBY read alignment files are also supported starting with IGV 2.0.

Both BAM and SAM files are described on the Samtools project page http://samtools.sourceforge.net/.

IGV requires that the alignment file, whether BAM or SAM, is indexed by coordinates. Indexing produces a secondary file with either a BAI or SAI extension, respectively. Place both the alignment file and index file in the same folder. When you specify the location of the alignment file, IGV automatically searches for the index file within the same directory.

  • The index files must have the same file name and must reside in the same directory as the file that it indexes.
    • For example, the index file for test-xyz.bam would be named test-xyz.bam.bai.
  • The GenePattern module Picard.SortSam will index both BAM and SAM files.
    • Select coordinate sort order and BAM output format to produce IGV-compatible sorted BAM file and BAI index file.
    • Igvtools does not process BAM files as alternative resources such as Samtools have been historically available.
  • If for some reason you choose to use a SAM alignment file directly on IGV,
    • the igvtools utility can both sort and index the file;
    • if the file is already sorted, an index will be automatically created upon first loading the file. IGV will attempt to store the index in the directory where the alignment file resides with an SAI extension. If that fails, the index is stored in the user's IGV directory.
    • The generated SAI index file is specific to IGV and is not compatible with Samtools or other applications.

Besides BAM and SAM, additional supported file formats related to alignments include ALIGNED, VCF, PSL, BED, and TDF.

Alignments Preferences

To display the Alignments Preferences window, click View>Preferences, then click the Alignments tab. For more detailed information about each of the preference options in this window, see the Alignment Preferences information in the User Interface section of the User Guide.

Alignments Panel

IGV displays sequence alignment data in a separate Alignments panel. The display changes as you zoom in. When zoomed out, IGV displays only coverage data, when available (more detail below).

When zoomed in to the alignment read visibility threshold (by default, 30 KB), IGV shows the reads. At this resolution the colored bars in the coverage track identify loci where more than 20% of the quality weighted reads differ from the reference.  For paired end alignments, reads that have a different than expected insert size are color coded to indicate the difference, and reads that have a mate on another chromosome are color coded to indicate the chromosome of the mate pair. (more detail below).

At higher resolutions read bases that do not match the reference are color coded, and insertions () and deletions relative to the reference become visible.  By default, read bases that match are displayed in gray.  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.  In addition, mismatched bases are assigned a transparency value proportional to the  read quality (phred) score.  This has the effect of de-emphasizing  low quality reads.  Transparency shading can be turned off temporarily from the pop-up menu, or persistently from the Preferences window.

By default, when zoomed in sufficiently, IGV displays a line at the center of the display. 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.

This option, along with many others, can be modified on the Alignments tab of the Preferences window.

Note that alignments that are displayed with light gray borders and white fill, as shown in the following screenshot, have a mapping quality equal to zero.  This value (0) means the read could also be mapped to another location.

Paired-End Alignments

IGV provides several options for working with paired-end alignments:

IGV colors paired-end alignments whose inferred insert size is larger than expected or whose mate read is aligned to a different chromosome. In addition, IGV colors paired-end alignments by pair orientation.

  • Details on Color by insert size are here.
    • 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.
  • Details on Color by pair orientation are here.
    • Shades of green to blue show structural events of inversions, duplications, and translocations.
  • Other Color by options are described here.

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 both insert size and pair orientation option.

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. Note: 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 above.
  • 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 a read to view information about the read, including the location of its paired mate.

 

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.

 

Return to Normal View

To return to the “normal view”, double-click the name panel at the top of one of the panes, or right-click in a name panel and select Switch to standard view.

 

View As Pairs

NGS paired-end reads can be drawn as a single "template", with a connecting line joining the two pairs.  To display this view, right-click over the alignments and select View as pairs.   If the alignment track does not contain paired-end data this option will not be available.

Example: 

Normal  view:

Paired view:

 

Insertions

In a gapped alignment, IGV indicates insertions with respect to the reference with a purple bar ().  Hover over the insertion symbol to view the inserted bases.

 

Deletions

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

Read Coverage

Default Coverage Data

IGV supplements each alignment track with a coverage track. When IGV is zoomed in sufficiently to display alignments, the coverage track displays the depth of the reads displayed at each locus as a grey 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).

To hide the coverage track, clear the Show Coverage Track option on the Alignments tab of the Preferences window.

Extended Coverage Data

To display coverage data at the whole genome or chromosome level, use the igvtools package (count command) to generate coverage data for the alignments file.  The resulting file can be associated with the alignment track by file naming convention, or loaded independently as a separate track.  

To associate a coverage track using filename, the track must be named as follows, and placed in the same directory as the alignment track:

<alignment file name>.tdf

For example,  the coverage track for test.bam would be named test.bam.tdf. 

To dynamically associate coverage data with a BAM track, choose the "Load Coverage Data" from either the alignment or coverage track pop-up menu.   When the alignment data is loaded with its matching coverage data, the coverage track displays data at all zoom levels.

Sorting Alignments

Alignments can be sorted by start location, strand, nucleotide, mapping quality, sample, or read group.

To sort alignments:

  1. Right-click a track to display the pop-up menu.
  2. Select a Sort option from the pop-up menu. IGV sorts the alignments that intersect the center line of the display.

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.

Illumina Sequencing Support

IGV includes limited support for viewing alignments in the "sorted.txt" format from the Illumina Pipeline version 1.3, with the following restrictions.

  • The contig fields (columns 12 and 19) are not supported
  • The Match chromosome field (column 11) must either be the name of a chromosome in an IGV genome, or an entry in the seqname to chromosome mapping file defined below.

Mapping File: To view alignments from a sorted.txt file in which the chromosome names are not chromosome names in IGV, a mapping file must be provided. This is a 2-column tab-delimited file with chromosome names from the sorted.txt file in column 1, and corresponding IGV chromosome names in column 2. The file must be named "sequence.map" and placed in a specific directory, which is platform dependent:

Windows: <user home>/igv/sam
Linux: <user home>/igv/sam
Mac: <user home>/.igv/sam

On Windows computers the user home directory is normally found at:

C:/Documents and Settings/<user name>