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# Copyright (c) 2016, Yu Hu and Mingyao Li (Department of Biostatistics and Epidemiology, University of Pennsylvania Perelman School of Medicine) #
# All rights reserved.                                                                                                                            #
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# PennDiff Tutorials #
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REQUIREMENTS:

    • Python v2.6+ 

    • R with packages "gcmr" and "betareg" installed

USAGE:
	python PennDiff.py -ref <RefSeqAnnotation> -est <isoformRelativeAbundances>

REQUIRED OPTIONS:
	 
	 -ref ---RefSeqAnnotation file can be downloaded from UCSC Genome Browsers. The file "RefSeqAnnotation_ECE1_example" is an example of RefSeqAnnotation file

RefSeqAnnotation_ECE1_example:

749     NM_001397       chr1    -       21543739        21616982        21546447        21616907        19      21543739,21548239,21551742,21553651,21554423,21560050,21562342,21563238,21564626,21571481,21573713,21582439,21584017,21585185,21586763,21599191,21605683,21616562,21616856,   21546624,21548335,21551933,21553719,21554534,21560154,21562420,21563337,21564737,21571596,21573856,21582631,21584083,21585332,21586885,21599404,21605825,21616649,21616982,   0       ECE1    cmpl    cmpl    0,0,1,2,2,0,0,0,0,2,0,0,0,0,1,1,0,0,0,
93      NM_001113348    chr1    -       21543739        21672034        21546447        21671871        19      21543739,21548239,21551742,21553651,21554423,21560050,21562342,21563238,21564626,21571481,21573713,21582439,21584017,21585185,21586763,21599191,21605683,21616562,21671868,   21546624,21548335,21551933,21553719,21554534,21560154,21562420,21563337,21564737,21571596,21573856,21582631,21584083,21585332,21586885,21599404,21605825,21616649,21672034,   0       ECE1    cmpl    cmpl    0,0,1,2,2,0,0,0,0,2,0,0,0,0,1,1,0,0,0,
749     NM_001113349    chr1    -       21543739        21616766        21546447        21616691        18      21543739,21548239,21551742,21553651,21554423,21560050,21562342,21563238,21564626,21571481,21573713,21582439,21584017,21585185,21586763,21599191,21605683,21616562,    21546624,21548335,21551933,21553719,21554534,21560154,21562420,21563337,21564737,21571596,21573856,21582631,21584083,21585332,21586885,21599404,21605825,21616766,    0       ECE1    cmpl    cmpl    0,0,1,2,2,0,0,0,0,2,0,0,0,0,1,1,0,0,
749     NM_001113347    chr1    -       21543739        21606183        21546447        21605927        17      21543739,21548239,21551742,21553651,21554423,21560050,21562342,21563238,21564626,21571481,21573713,21582439,21584017,21585185,21586763,21599191,21605683,     21546624,21548335,21551933,21553719,21554534,21560154,21562420,21563337,21564737,21571596,21573856,21582631,21584083,21585332,21586885,21599404,21606183,     0       ECE1    cmpl    cmpl    0,0,1,2,2,0,0,0,0,2,0,0,0,0,1,1,0,


	 -est ---Isoform relative abundances can be obtained by using existing software such as PennSeq or Cufflinks. However, the format of results is consistent across different software.Thus we ask user to give a isoformRelativeAbundances file with format as example file "isoformRelativeAbundances_ECE1_example" shows:

	      • Column 1: gene name
	      • Column 2: isoform name
	      • Column 3: sample number
	      • Column 4: estimated isoform relative abundances
	      • Column 5: condition group (1 or 2)

isoformRelativeAbundances_ECE1_example:

ECE1    NM_001113347    0       0.257339787046751       1
ECE1    NM_001113349    0       0.0899339615416256      1
ECE1    NM_001113348    0       0.608750982445272       1
ECE1    NM_001397       0       0.0439752689663516      1
ECE1    NM_001113347    1       0.294457146326302       1
ECE1    NM_001113349    1       0.087303489040162       1
ECE1    NM_001113348    1       0.571130340575692       1
ECE1    NM_001397       1       0.047109024057844       1


OUTPUT:
	"geneDASResults" file: Gene-based test results file with 3 columns:
		• Column 1: gene name
		• Column 2: test P-Value
		• Column 3: Hellinger distance which measures the splicing difference between two conditions for a gene in terms of isoform relative abundances
		
	"exonDASResults" file: Exon-based test results file with 10 columns:
		• Column 1: gene name
		• Column 2: DAS testing group where exons within same group share same P-Value
		• Column 3: exon location
		• Column 4: test P-Value
		• Column 5: exon inclusion level difference which measures splicing difference between two conditions for an spliced exon
		• Column 6: "+"/"-" if exon inclusion level of condition 1 is greater/smaller than condition 2.
		• Column 7: number of samples in condition 1 group
		• Column 8: exon inclusion levels of each sample in condition 1 group
		• Column 9: number of samples in condition 2 group
		• Column 10: exon inclusion levels of each sample in condition 2 group

	
CONTACT INFORMATION:
For more information, such as bug reports and comments, please contact Yu Hu via huyu1@mail.med.upenn.edu.