4.1. Workaround if you do not have biological replicates (version 1.*, 2.0 and 2.0.1)

If you do not have biological replicates for your samples, here is a workaround (the strategy is to simulate a biological read count file of the junctions based on the real dataset, in each condition):

1. Run everything as instructed in the manual until finishing JUM_2-3.sh (completing Step 7). During the process:

   • When running JUM_2-2.sh, set the --Filenum parameter to be 1. for example:

     $ cd control
     $ bash /user/home/JUM_2.0/JUM_2-2.sh --Folder /user/home/JUM_2.0 --Threshold 10 --Filenum 1 --Condition control
     $ cd ..
     $ cd treatment
     $ bash /user/home/JUM_2.0/JUM_2-2.sh --Folder /user/home/JUM_2.0 --Threshold 10 --Filenum 1 --Condition treatment
     $ cd ..

   • When running JUM_2-3.sh, set the --Filenum parameter to be 1. for example:

     $ bash /user/home/JUM_2.0/JUM_2-3.sh --Folder /user/home/JUM_2.0 --Threshold 10 --Filenum 1 --IRthreshold 10 --Readlength 100 --Thread 3

   NOTES

   Since you only have one sample under each condition, it is recommended to be more stringent about junction filtering in JUM_2-2.sh, as you do not have extra replicates to provide buffer for junction quality reassurance. For example, here we set --Threshold to be 10 instead of 5, so as to make sure the junctions passing to JUM are real biological ones but not random noise. You can set this number to be even higher, based on your datasets.

2. Enter the JUM_diff/ folder, and before you run the Rscript:

   • Run the script vary_for_replicate.pl in the JUM package as follows:

    $ perl /user/home/JUM_1.3.12/vary_for_replicate.pl ctrl_combined_count.txt > temp_ctrl_count.txt
    $ perl /user/home/JUM_1.3.12/vary_for_replicate.pl treat_combined_count.txt > temp_treat_count.txt
    $ less temp_ctrl_count.txt | cut -f1,3 > ctrlRep_combined_count.txt
    $ less temp_treat_count.txt | cut -f1,3 > treatRep_combined_count.txt
    $ rm temp_ctrl_count.txt
    $ rm temp_treat_count.txt

   • Continue with the instructions in Step 8 in the manual. You need to edit the experiment_design.txt file as follows:

         Condition
   ctrl control
   ctrlRep control
   treat treatment
   treatRep treatment

3. Continue running the steps as instructed in the manual. During the process:

   • When running JUM_3.sh, set the --Totalnum parameter to be the total # of samples including the simulated ones. In the example here, 4.

   • When running JUM_3.sh, set the --Smallnum parameter to be the # of actual biological control/treated samples, whichever is smaller. In the example here, 1.

     for example:

     $ bash /user/home/JUM_2.0/JUM_3.sh --Folder /user/home/JUM_2.0 --Test pvalue --Cutoff 0.05 --Totalnum 4 --Smallnum 1

   • When running JUM_4.sh, set the --condition1num parameter to be the total # of control samples including the simulated ones. In the example here, 2.

   • When running JUM_4.sh, set the --condition2num parameter to be the total # of treated samples including the simulated ones. In the example here, 2.

     for example:

     bash /user/home/JUM_2.0/JUM_4.sh --Folder /user/home/JUM_2.0 --Test pvalue --Cutoff 0.05 --condition1num 2 --condition2num 2 --REF refFlat.txt