SGSGeneLoss

What does SGSGeneLoss depend on?

SGSGeneLoss depends on the following:

• Java 1.6 or higher
• R/3.1.0
• picard-tools
• ggplot2
• ggbio

Download

• Latest Version 0.1 (29/04/2014):
  • SGSGeneLoss.v0.1.tar.gz should contain
    • three main programs: SGSGeneLoss.v0.1.jar, graph_chromosomes.v0.1.R, graph_circles.v0.1.R
    • readme file
    • folder with source code

From now on in this manual SGSGeneLoss.v0.1.jar, graph_chromosomes.v0.1.R, graph_circles.v0.1.R are referred to as SGSGeneLoss.jar, graph_chromosomes.R, graph_circles.R

To run the programs you have to use full names SGSGeneLoss.v0.1.jar, graph_chromosomes.v0.1.R, graph_circles.v0.1.R

How to install?

• SGSGeneLoss.tar.gz
• Unpack SGSGeneLoss.tar.gz and place SGSGeneLoss.jar and all the R scripts in chosen directory/directories, for example ./my_geneloss
• Move into ./my_geneloss and create SGSGeneLoss_lib directory (on linux: cd ./my_geneloss, mkdir SGSGeneLoss_lib directory)
  • The name of the lib directory is the name of the .jar file without .jar extension + _lib, so if you are using SGSGeneLoss.v0.1.jar the lib directory is: SGSGeneLoss.v0.1_lib
• Download picard-tools (SGSGeneLoss was tested with picard-tools 1.89)
• Place picard-1.89.jar and sam-1.89.jar in ./my_geneloss/SGSGeneLoss_lib
• Now you are ready to run SGSGeneLoss

Input and output files for SGSGeneLoss.jar

• Input files:
  • Sorted, indexed .bam file with sequencing reads mapped to the reference genome sequence, multiple .bam files can be provided as comma separated list
  • Gff3 file with reference genome annotation, has to contain gene, mRNA and exon fields
• Output files
  • Result files for each chromosome separately - .excov
  • File with overall stats - stats.txt
  • File with summary for all the chromosomes used - chrs.csv (this file is used by one of the R scripts)
  • File with list of genes lost for all the chromosomes - graph.csv (this file is used by one of the R scripts)

Command line options for SGSGeneLoss.jar

Required:

bamPath - path to your bam file/files, has to end with / or \ bamPath=/home/my_bams/

bamFileList - a single .bam file or a comma separated list, only file names, bam and corresponding .bai files have to be in a directory provided in bamPath bamFileList=bam1.bam,bam2.bam

gffFile - location of gff3 file gffFile=/home/my_gffs/annot.gff3

outDirPath - location output directory, has to end with / or \ outDirPath=/home/my_results

Optional:

minCov - minimal coverage threshold to consider position covered [minCov=1]

chromosomeList - comma separated list of chromosomes to be used for analysis, use all, for all chromosomes [chromosomeList=all]

lostCutoff - coverage cutoff to consider gene as lost for calculating stats [lostCutoff=0.0]

covCats - coverage categories for visualization [cavCats=0,10,20,30,40,70]

extendedFmt - used extended format, additional info included in output files [regular format]

To see help run: java -jar SGSGeneLoss.jar help

Sample command

• Move into directory where SGSGeneLoss.jar is
• Please make sure that all your supplied paths end with / or \

java -Xmx4g -jar SGSGeneLoss.jar bamPath=/home/uqagnieszka/bams/ bamFileList=arabidopsis.sorted.bam gffFile=/home/gff_files/Athaliana_167_gene_exons.gff3 outDirPath=/home/uqagnieszka/results/
chromosomeList=all

java -Xmx4g -jar SGSGeneLoss.jar bamPath=/home/uqagnieszka/bams/ bamFileList=arabidopsis.sorted.bam, arabidopsis2.sorted.bam gffFile=/home/gff_files/Athaliana_167_gene_exons.gff3
outDirPath=/home/uqagnieszka/results/ chromosomeList=Chr1,Chr2 minCov=2 lostCutoff=0.05 covCats=0,2,5,10,20 extendedFmt

Output files format

All the output files are comma separated text files.

• .excov files - files with results for each chromosome (files use chromosome names as in .bam files), files come in two formats basic (default) or extended (extendedFmt)
  • basic format: chromosome,ID,is_lost,start_position,end_postion,frac_exons_covered,frac_gene_covered,ave_cov_depth_exons,cov_cat,ave_cove_depth_gene
  • extended format: contains additional columns with information about each of the exons
• stats.csv - file with summary information about all genes
• chrs.csv - file with summary information about chromosomes
  • chr,start,end,len
• graph.csv - file with list of genes lost as determined by lostCutoff
  • chr,id,start,end

Plotting results

Results are visualized using R scripts.

Two ways of visualization are possible:

• results per chromosome
• results for all chromosomes as a circular graph

Results per chromosome:

What you need:

• script graph_chromosomes.R
• .excov files (either basic or extended) with results from SGSGeneLoss.jar: Chr1.excov, Chr2.excov etc.
• directory (location) where files with results from SGSGeneLoss.jar: Chr1.excov, Chr2.excov etc. can be found

graph_chromosomes.R takes two arguments in this order:

1. location of directory where .excov file are located

2. gene loss cutoff

Rscript --vanilla graph_chromosomes.R /home/uqagnieszka/results 0.0

Summary results for all chromosomes, possibly multiple samples:

What you need:

• script graph_circles.R
• graph.csv from SGSGeneLoss.jar run
• chrs.csv from SGSGeneLoss.jar run
• file assigning numeric order to chromosomes (this is done because some chromosomes have complicated names and sorting in ASCII order does not always work) - file should look like this, chromosome names will be replaced by corresponding numbers

chrs,no
chr1,1
chr2,2
chr10,10

graph_circles.R takes four arguments in this order:

1. file with chromosome info - chrs.csv from SGSGeneLoss.jar run

2. file with chromosome order

3. file with genes lost - graph.csv from SGSGeneLoss.jar run; it can be a comma separated list of multiple files (for example multiple samples). Circles will be drawn in the following order:

first file in the list is the innermost circle, so if you have graph1.csv,graph2.csv,graph3.csv, order of circles will reflect order of files, starting from the inside

4. output file

Rscript --vanilla graph_circles.R chrs.csv chrs_order.csv graph1.csv,graph2.csv,graph3.csv out.png

FAQ

• If memory consumption is a problem please consider increasing -Xmx or splitting your .bam files

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