Friday, February 13, 2015

Running igvtools from the Command Line

Downloading igvtools
The igvtools utilities can be downloaded from the Downloads page on the IGV Web site.
          igvtools_<version #>.zip includes the jar file and shell scripts for running igvtools, as well as the genome files.
          igvtools_nogenomes_<version #>.zip includes the jar file and shell scripts and shell scripts for running igvtools.
Starting with shell scripts
The igvtools utilities can be invoked, with or without the graphical user interface (GUI), from one of the following scripts:
   igvtools (command-line version for linux and  Mac OS 10.x)
   igvtools_gui (gui version for linux and  Mac OS 10.x)
   igvtools.bat (command-line version for windows)
   igvtools_gui.bat (gui version for windows)
The general form of the command-line version is:
   igvtools [command] [options][arguments]
or
   igvtools.bat [command] [options][arguments]
Recognized commands, options, arguments, and file types are described below.
Starting with java
Igvtools can also be started directly using Java.  This option allows more control over Java parameters, such as the maximum memory to allocate.  In this example, igvtools is started with 1500 MB of memory allocated:
   java -Xmx1500m  -Djava.awt.headless=true -jar igvtools.jar [command] [options][arguments]
To start with a GUI the command is
   java -Xmx1500m  -jar igvtools.jar -g
Memory settings
The scripts above allocate a fixed amount of memory.  If this amount is not available on your platform you will get an error along the lines of "Could not start the Virtual Machine".   If this happens you will need to edit the scripts to reduce the amount of memory requested,  or use the Java startup option.  The memory is set via a "-Xmx" parameter. For example  -Xmx1500m requests 1500 MB,  -Xmx1g requests 1 gigabyte.
Genome
The genome argument in the toTDF and count command can be either an id, or a full path to a .chrom.sizes or an IGV .genome file.

Commands

toTDF

The toTDF command converts a sorted data input file to a binary tiled data (.tdf) file. Use this command to pre-process large datasets for improved IGV performance. 
Supported input file formats are: .wig, .cn, .snp, .igv, and .gct.
Note: This tool was previously known as "tile"
Usage:
          igvtools toTDF [options]  [inputFile] [outputFile] [genome]
Required arguments:
          inputFile    The input file (see supported formats below).
          outputFile   Binary output file.  Must end in ".tdf".
          genome      A genome id or path to a .chrom.sizes or .genome file.  Default is hg18.
Options:
  -z num  Specifies the maximum zoom level to precompute. The default
               value is 7 and is sufficient for most files. To reduce file
               size at the expense of IGV performance this value can be
               reduced.
  -f  list     A comma delimited list specifying window functions to use
               when reducing the data to precomputed tiles.   Possible
               values are min, max, and mean.  By default only the mean
               is calculated.
  -p file    Specifies a "bed" file to be used to map probe identifiers
               to locations.  This option is useful when preprocessing . gct
               files.  The bed file should contain 4 columns:
                           chr start end name
               where name is the probe name in the .gct file.
Example:
          igvtools toTDF -z 5  copyNumberFile.cn copyNumberFile.tdf hg18
Notes:
Data file formats, with the exception of .gct files, must be sorted by start position.  Files can be sorted with the sortcommand described below.  Attempting to preprocess an unsorted file will result in an error.

Count

The count command computes average feature density over a specified window size across the genome. Common usages include computing coverage for alignment files and counting hits in Chip-seq experiments. By default, the resulting file will be displayed as a bar chart when loaded into IGV.
Supported input file formats are: .sam, .bam, .aligned, .psl, .pslx, and .bed.
Usage:
          igvtools count [options] [inputFile] [outputFile] [genome]
Required arguments:
          inputFile    The input file (see supported formats above).
          outputFile   The output file, which can be binary "tdf" or ascii "wig format.  The filename must.  The filename must end in ".tdf" or ".wig", or be the special string "stdout".  To indicate that you want to output both a .tdf and a .wig file, list both output filenames as a single string,  separated by a comma with no other delimiters. If the output file is named "stdout" the output will be written to the standard output stream in wig format.
          genome      A genome id or path to a .chrom.sizes or .genome file.  Default is hg18.
Options:
-z, --maxZoom num
Specifies the maximum zoom level to precompute.
-w, --windowSize num
The window size over which coverage is averaged. Defaults to 25 bp.
 -e, --extFactor num
The read or feature is extended by the specified distance in bp prior to counting. This option is useful for chip-seq and rna-seq applications. The value is generally set to the average fragment length of the library minus the average read length.
--preExtFactor num
The read is extended upstream from the 5' end by the specified distance.
--postExtFactor num
Effectively overrides the read length, defines the downstream extent from the 5' end. Intended for use with preExtFactor.
-f, --windowFunctions list
A comma delimited list specifying window functions to use when reducing the data to precomputed tiles. Possible values are min, max, mean, median, p2, p10, p90, and p98. The "p" values represent percentile, so p2=2nd percentile, etc.
--strands [arg]
By default, counting is combined among both strands. This setting outputs the count for each strand separately. Legal argument values are 'read' or 'first'. 'read' Separates count by 'read' strand, 'first' uses the first in pair strand".  Results are saved in a separate column for .wig output, and a separate track for TDF output.
--bases
Count the occurrence of each base (A,G,C,T,N). Takes no arguments. Results are saved in a separate column for .wig output, and a separate track for TDF output.
--query [querystring]
Only count a specific region. Query string has syntax <chr>:<start>-<end>. e.g. chr1:100-1000. Input file must be indexed. 
--minMapQuality [mqual]
Set the minimum mapping quality of reads to include. Default is 0.
--includeDuplicates
Include duplicate alignments in count. Default false. If this flag is included, duplicates are counted. Takes no arguments
--pairs
Compute coverage from paired alignments counting the entire insert as covered. When using this option only reads marked "proper pairs" are used.
Notes:
The input file must be sorted by start position. See the sort command below.
Example:
          igvtools count -z 5 -w 25 -e 250 alignments.bam  alignments.cov.tdf  hg18

Index

Creates an index for an alignment or  feature file.  Index files are required for loading alignment files into IGV, and can significantly improve performance for large feature files.  Note that the index file is not directly loaded into IGV. Rather, IGV looks for the index file when the alignment or feature file is loaded.  This command does not take an output file argument. Instead, the filename is generated by appending ".sai" (for alignments) or ".idx" (for features) to the input filename as IGV relies on this naming convention to find the index . The input file must be sorted by start position (see sort command, below). 
Supported input file formats are: .sam, .aligned, .vcf, .psl, and .bed.
NOTES:
  • This command will not index a binary (BAM) file.  Use the samtools package to sort and index BAM files. 
  • The "sai" index is an IGV format, it does not work with samtools or any other application.
Usage:
  igvtools index [inputFile]

Sort

Sorts the input file by start position, as required.
Supported input file formats are: .cn, .igv, .sam, .aligned, .psl, .bed, and .vcf.
NOTE:  This command does not work with BAM files.  The samtools package can be used to sort .bam files.
Usage:
          igvtools  sort [options] [inputFile]  [outputFile]
Required arguments:
          inputFile 
          outputFile 
The special string "stdout" can be used as [outputFile], in which case the output will
be written to the standard output stream instead of a file.
Options:
  -t tmpdir  Specify a temporary working directory.  For large input files
             this directory will be used to store intermediate results of
             the sort. The default is the users temp directory.
  -m maxRecords  The maximum number of records to keep in memory during the
             sort.  The default value is 500000.  Increase this number
             if you receive "too many open files" errors.   Decrease it
             if you experience "out of memory" errors.

Version

  Prints the igvtools version number to the console.

http://www.broadinstitute.org/igv/igvtools_commandline

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