ASD

 ASD(Alternative splicing detector) is a user-friendly software that aims to recognize and detect differential alternative splicing(AS) exons in two samples of RNA-Seq data. Since AS events always have two obvious feature in RNA-Seq data, which are altered junction reads and altered coverage of AS-exons, the algorithm of this software considers these two factors while calculating a combine P-value to investigate whether a AS event is statistically significant or not.

Reference: Zhou X, et al. Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation. Nucleic Acids Res. 2014 Apr;42(6):4019-30.

 ASD is a java-based software. Thus you need to install jre-1.7 first. Then you need download ASD and Assigned 755 permisssion to the ASDrun.sh if you're running ASD in linux. You would like to use the command:

sudo chmod 755 /your/path/to/ASD/ASDrun.sh

to install this

 To start ASD, you just need to double click ASDrun.sh in linux or ASDrun.bat in windows. ASD need bam version file that is generated by TopHat or any other RNA-Seq maping softwares which contains the junction reads, as well as a GTF version file corresponding to the genome you use to do the mapping. Optionally, If you want to get the sequence near the splicing site, you would need to provide the genome sequence fold to ASD.

1. Click button AddBam to load in the bam format files generated from the mapping software such as tophat. Fill the prefix column and group columns. Bam files come from same technical replicates should with same prefix names, and with same group names if they come form same biological replicates.
2. Click OpenGTF to load in the GTF version files that records the information about reconstructed transcripts.
3. Click AddCmp and then put down names of both control and treated groups, which should be consistent with those that were used at step 1.
4. It is possible that there are more than one alternative splicing locations in a single gene. With "Display All Splicing Events" chosen, we could get all the alternative splicing events. With "Display All Splicing Events" not chosen,we could only get the alternative splicing events that are statistically significant.
5. Click OpenSeqPath to load the folder recoding the whole genome sequences into the platform. Users need to notice that there is only one file for a single chromosome.
6. Click SaveTO to set up the saving path,After the above settings, the ASD UI should like this:
   
7. Click runPASH to start the detection for significant alternative splicing events, and now you can have a rest and take a cup of coffee.
8. Result format
   

   AccID	Location	Treat_JuncInfo	Col_JuncInfo	TreatCoverage	ColCoverage	P-Value	SplicingType	Description
   Foxp1	chr6:99028164-99028258	190::85	45::67	160.0::211.0	10.0::147.0	1.22E-07	altstart	forkhead box P1
   Immt	chr6:71853173-71853268	248::54	163::256	31.0::175.0	121.0::180.0	1.80E-05	cassette	Immt protein
   Mars	chr10:127296717-127296740	212::34	269::0	20.0::137.0	1.0::130.0	2.42E-05	cassette	methionine-tRNA synthetase
   Raly	chr2:154821007-154821089	648::943	86::387	406.0::324.0	193.0::47.0	3.31E-05	cassette	RNA-binding protein Raly
   Hmga1	chr17:27556952-27557066	227::60	200::7	57.0::313.0	6.0::330.0	4.71E-05	cassette	high mobility group AT-hook 1
   Tbl1x	chrx:77584794-77584859	21::39	24::1	22.0::167.0	1.0::170.0	5.22E-05	cassette	transducin (beta)-like 1 X-linked

   
   

   AccID:	the gene Access ID or Symbol, depends on the information provide by GTF file.
   Location:	Splicing events happened location, canbe enter in IGV to see splicing details.
   Treat_JuncInfo:	Junction reads number information in treat sample.
   Col_JuncInfo:	Junction reads number information in control sample.
   TreatCoverage:	Treatment splicing exon coverage and gene coverage
   ColCoverage:	Control splicing exon coverage and gene coverage
   P-value:	The significant of differential splicing event.
   SplicingType:	Type of this alternative splicing site.
   Description:	Desciption of this gene, depends on the information provide by GTF file.

9. About GTF format
   the GTF file format should follow two rules:
   1. No matter the strand of a gene, the location of the exon should be sorted from low to high,
   2. If there is a CDS, it should be wroten after all the exons involved in.
   just like the table below:

   chr1	novelbio	exon	865535	865716	.	+	.	gene_id "SAMD11"; transcript_id "NM_152486"; gene_name "SAMD11";
   chr1	novelbio	CDS	865535	865716	.	+	.	gene_id "SAMD11"; transcript_id "NM_152486"; gene_name "SAMD11";
   chr1	novelbio	exon	866419	866469	.	+	.	gene_id "SAMD11"; transcript_id "NM_152486"; gene_name "SAMD11";
   chr1	novelbio	CDS	866419	866469	.	+	.	gene_id "SAMD11"; transcript_id "NM_152486"; gene_name "SAMD11";
   chr1	novelbio	exon	879583	880180	.	-	.	gene_id "NOC2L"; transcript_id "NM_015658"; gene_name "NOC2L";
   chr1	novelbio	CDS	880077	880180	.	-	.	gene_id "NOC2L"; transcript_id "NM_015658"; gene_name "NOC2L"; p_id "P5581"; tss_id "TSS17475";
   chr1	novelbio	exon	880437	880526	.	-	.	gene_id "NOC2L"; transcript_id "NM_015658"; gene_name "NOC2L"; p_id "P5581"; tss_id "TSS17475";
   chr1	novelbio	CDS	880437	880526	.	-	.	gene_id "NOC2L"; transcript_id "NM_015658"; gene_name "NOC2L"; p_id "P5581"; tss_id "TSS17475";

   Alse you can only have exon infomation without CDS information, because ASD extract the junction infomation from exon lines.

 This version fix bugs and optimize the transcriptome reconstruct process. And can now support Gff3 files download from NCBI( the Human, Mouse and Rat Gff3 have already been tested).
2014-10-10

 This version fix bugs and add a function to reconstruct transcriptome, and can detect splicing events even not include in gff file.
2014-04-18

 This is the first Version!
2013-07-18