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Name : FusionScan
Version : 1.0
Last Updated : 2014-10-25
Description : FusionScan is a accurate prediction tool for fusion genes from RNA-seq data.
FusionScan has better precision and recall than other tools.
Please see http://fusionscan.ewha.ac.kr/ for FusionScan manual and documentation.
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DOWNLOAD
GETTING THE SOFTWARE AND INDEX FILES
: You can get the recent version of fusionscan from https://github.com/iamlife/FusionScan or under link.
FILE NAME DESCRIPTION RECOMENDED LOCATION
fusionscan fusionscan src files your wanted location
tar xvfz fusionscan.tar.gz
cd fusionscan/
hg19.2bit.gz hg19.2bit file to run gfServer(blat) /fusionscan/src_dir/
nib_files_hg19.tar.gz hg19 nib files to use nibFrag /fusionscan/src_dir/
bowtie2_index_hg19.tar.gz bowtie2 index files for hg19 & hg19.fa /fusionscan/src_dir/
bowtie2_index_refMrna.tar.gz bowtie2 index files for refMrna & refMrna.fa /fusionscan/src_dir/
ssaha2_index_hg19.tar.gz ssaha2 index files for hg19 /fusionscan/src_dir/
gene_dir.tar.gz gene annotation files /fusionscan/
rmsk_dir.tar.gz repeatMasker files /fusionscan/
REQUIREMENT
ENVIROMENT TO RUN THE SOFTWARE
- JDK1.7 or Jre1.7, Python
: FusionScan's main process is developed with JAVA language and whole-process is in Python script.
REQUIRED INDEX AND SEQUENCE FILES
- bowtie2 index files for hg19 & hg19.fa
- bowtie2 index files for refMrna & refMrna.fa
- ssaha2 index files for hg19
REQUIRED PROGRAMS (go to SETTING)
- bowtie2: to obtain unmapped reads
- SSAHA2: to align reads to genome
- BLAT (gfServer, gfClient): to confirm final candidates
- nibFrag: to make pseudo fusion transcript
- bl2seq (blast): to search sequence homology
- fastq_quality_trimmer, fastx_collapser, fastx_artifacts_filter (FASTX-Toolkit): to do pre-processes
HOW TO RUN
INSTALL
$ tar -xvzf fusionscan.tar.gz
$ cd fusionscan/
*** each directory includes ***
- fusionscan/ :
- Run_FusionScan.py : to run whole process
- fusionscan/src_dir/: contains various programs which are used for
sequence alignment, sequence comparison, getting sequences from genome.
- FusionScan.jar: FusionScan jar file
* Under files should be downloaded in src_dir/
- hg19.2bit: to use gfServer
- nib_files_hg19/* : to use nibFrag
- bowtie2_index_hg19/*: to use BOWTIE2
- bowtie2_index_refMrna/*: to use BOWTIE2
- ssaha2_index_hg19/*: to use SSAHA2
* Under binary src files which are proper to your system should be copied in src_dir/
in case of linux_ix86_64, all binaries are included in fusionscan/src_dir/
- nibFrag: from BLAT
- gfClient: from BLAT
- gfServer: from BLAT
- bl2seq: from BLAST
- bowtie2: from BOWTIE2
- bowtie2-align: from BOWTIE2
- bowtie2-align-debug: from BOWTIE2
- fastq_quality_trimmer: from FASTX-Toolkit
- fastx_collapser: from FASTX-Toolkit
- fastx_artifacts_filter: from FASTX-Toolkit
- ssaha2: from SSAHA2
- ssaha2Build: from SSAHA2
- fusionscan/gene_dir/ : contains various gene annotation files.
These files can be downloaded from here and extract it in fusionscan/
- duplicated_gene_database_dgd_Hsa_all_v68.tsv: duplicated gene database file
- pseudogeneHUGO.txt: HGUO file's pseudogene part info file
- refGene_and_indexes.txt: indexed refGene file
- refGene.txt: raw refGene file
- fusionscan/rmsk_dir/ : contains simple formatted repeatMasker data.
These files can be downloaded from here and extract it in fusionscan/
SETTING
0. download index files and fasta files for hg19 and refMrna from here and extract it in fusionscan/
*** FusionScan needs bowtie2, SSAHA2, bl2seq and FASTX-Toolkit binary files.
1. Bowtie2 : http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.1.0/
Copy bowtie2's executable files (bowtie2, bowtie2-align, bowtie2-align-debug)
proper to your linux system to fusionscan/src_dir/
$ cp bowtie2 fusionscan/src_dir/
$ cp bowtie2-align fusionscan/src_dir/
$ cp bowtie2-align-debug fusionscan/src_dir/
2. SSAHA2 : http://www.sanger.ac.uk/resources/software/ssaha2/
Copy SSAHA2's executalbe files (ssaha2, ssaha2Build)
proper to your linux system to fusionscan/src_dir/
$ cp ssaha2 fusionscan/src_dir/
$ cp ssaha2Build fusionscan/src_dir/
3. FASTX-Toolkit : http://hannonlab.cshl.edu/fastx_toolkit/download.html
Copy FASTX-Toolkit's executable file (fastq_quality_trimmer, fastx_collapser, fastx_artifacts_filter)
proper to your linux system to fusionscan/src_dir/
$ cp fastq_quality_trimmer fusionscan/src_dir/
$ cp fastx_collapser fusionscan/src_dir/
$ cp fastx_artifacts_filter fusionscan/src_dir/
4. blast (bl2seq) : ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/LATEST/
Copy blast's executable file (bl2seq)
proper to your linux system to fusionscan/src_dir/
$ cp bl2seq fusionscan/src_dir/
5. blat : http://hgdownload.cse.ucsc.edu/admin/exe/
Copy blat's executable file (gfClient, gfServer, nibFrag)
proper to your linux system to fusionscan/src_dir/
$ cp gfClient fusionscan/src_dir/
$ cp gfServer fusionscan/src_dir/
$ cp nibFrag fusionscan/src_dir/
*** cf1. all index files for two alignment program are included in fusionscan/src_dir/)
*** cf2. in case of linux_ix86_64 : all binaries are included in fusionscan/src_dir/
COMMANDS
Usage:
python Run_FusionScan.py <reads1[,reads2]> <output-dir> <read-length> <gfserver host:port> [options]
** <gfserver host:port> is need to use blat (gfclient) at the last step.
Options:
--phred33 : qualities are Phred+33 [ default]
--phred64 : qualities are Phred+64
-P/--threads : number of threads <int> [ default: 1]
-ms/--min-seed : minimum number of seed reads <int> [ default: 2]
-md/--min-distance : minimum distance between two genes <int> [ default: 100000 ]
-kfgs/--known-fusion-gene-search : searching known kinase contained fusion genes
Example:
python Run_FusionScan.py Test.fa Test 75 00.00.00.00:8100 --phred33 -P 1 -ms 2
OUTPUTS
1. FUSION INFORMATION FILE
fusionscan/project_name/all_candidates_for_project_name_seed2.txt
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| column | explanation | example |
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| Hgene | head gene symbol | EML4 |
| Hchr | head gene's chromosome | chr2 |
| Hstrand | head gene's strnad | + |
| Hbp | head gene's break point | 42522656 |
| Tgene | tail gene symbol | ALK |
| Tchr | tail gene's chromosome | chr2 |
| Tstrand | tail gene's strand | - |
| Tbp | tail gene's break point | 29446394 |
| Hg-Tg | head-tail gene pair | EML4-ALK |
| # of seed read | seed read count | 13 |
| BPseq | 60bp sequence before & after of exon junction break point | TAGAGCCCACACCTGGGAAAGGACCTAAAGtgtaccgccggaagcaccaggagctgcaag |
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2. READ ALIGNMENT FILE FOR EACH FUSION CANDIDATE
fusionscan/project_name/alignment_dir/EML4_ALK.RNAalign
3. CREATED DIRECTORIES
- fusionscan/project_name/: is created when you run Run_FusionScan.py.
under all directories and files are created in project_name directory.
- alignment_dir/ (*.RNAalign*): contains the alignment files for fusion candidate reads,
these are created when the total analysis is end.
- fa_dir/: contains the splited fasta files.
- pslx_dir/: contains the pslx formated alignment files.
- log_dir/: contains the log files.
- all_candidates_for_project_name_seed1.txt: result report for all candidates.
- all_candidates_for_project_name_seed2.txt: result report for candidates having more than 2 seed reads.
- cout_per_each_step_project_name: statistics for each step.
- log_project_name: logs for each reads during running FusionScan.jar
4. COVERAGE PLOT FOR FUSON GENE
Usage: python make_cvg_plot.py <FusionScan_result_file> <RNA_seq_bamfile>
** FusionScan's result as same as upper format , especially cases seed >= 2
** RNA-seq has to be aligned on hg19. This should be input by user.
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