...

1. Open a BAM file.
2. Zoom in on the gene or region of interest.
3. Click Load Data to load sequence read alignments into the new track. 

To run FindJunctions:

1. Right-click your data's track label.
2. Select Track Operations > FindJunctions or Track Operations > > FindJunctions (TopHat). 
3. Enter a value or use the default. At least this many bases must align across a putative intron for a read to be counted as support for a junction.
4. Select OK to run FindJunctions.

A Image Added

Did it work? 

If yes, a new track will then appear containing junction features bracketing introns (see below). Labels report the number of spliced alignments that supported the junction. 

 Image Removed

Using FindJunctions from the command line

To run FindJunctions as a stand-alone program, visit https://bitbucket.org/lorainelab/findjunctions. Follow the instructions there to compile FindJunctions and create a "jar" file using Apache Maven.

FindJunctions takes one argument, the name of a BAM file, and multiple options:

• -u (unique) Ensures that only uniquely mapping spliced reads (with NH tag equal to 1) will be used to construct junctions. Default is to use all reads regardless of mapping quality or number of mappings obtained.
• -n Number of bases that must map to either side of a putative intron for a spliced read to be used to create or support a junction feature. Default is 5.
• -b Absolute path to a .2bit format genomic sequence file that will be used to identify junction strand. Required.
• -o (output) Name of the junctions file that will be written. Default is print to stdout.

Output is tab-delimited BED12 format. The name field contains a name constructed from the location of the junction and the score field contains the number of spliced alignments supporting each junction. 

Example:

java -Xmx1g -jar FindJunction_exe.jar -u -n 5 -b Genome.2bit -o FJ.bed sample1.bam,sample2.bam

Here, the -Xmx1g option specifies that the program can run with up to 1 Gb of computer memory (RAM) using the code in jar file (-jar) FindJunction_exe.jarEach inferred intron appears as a thin line connecting two blocks, one on either side of the line. The width of these "flanking" blocks indicate the number of bases you entered in step 3 above, unless you selected the "Find Junctions (TopHat)" option. If you chose that option, then FindJunctions will create flanking blocks as large as the longest aligned region detected from any of the sequence read alignments. For example, if there was just one sequence that aligned across an inferred intron with 20 bases on either side of the intron, then the blocks will be 20 bases in size. The name "TopHat" comes from a software program with similar behavior. TopHat is an RNA-Seq sequence to genomic assembly sequence alignment tool that created junction feature files (called "junctions.bed") using sequence read alignments. For more information about TopHat, visit the TopHat Manual.

 Image Added

Using FindJunctions from the command line

To run FindJunctions as a stand-alone program, visit https://bitbucket.org/lorainelab/findjunctions.

Follow the instructions there to compile FindJunctions and run it.