This pipeline is used to simulate SC-RNA data.

All the SC-RNA data which simulated for DSJ-detection are based on Spanki RNA-seq reads simulator, which supports specific RPK (reads per kilo-base) value for each transcript. The dropout event is based brie simulator script with slight change.Thus,you need to install Spanki and brie first.

Following BRIE tutorial to install is advised.

BRIE tutorial
follow the brie tutorial and install brie successfully.Then download simuDropout_modify.pyscript to ./brie/simulator/ directory

Step1 simulate two original rpk files
perl sim_rpk.pl output_file_prefix gene_rpk degree_diff
To simulate a differential expression junction related gene,for every two transcript belonging to the same gene in a file,keep gene_rpk * gene_length=transcript1_rpk * transcript1_length + transcript2_rpk * transcript2_length
For A sample, transcript1_rpk/transcript2_rpk value is between degree_diff/2 and degree_diff. Keep the ratio of B inverse to A.

Example
perl sim_rpk.pl sim_rpk1 50 8
This commond outputs two files:sim_rpk1A sim_rpk1B
Step2 simulate fastq and bam files

  python Spanki-master/bin/spankisim_transcripts  
  -o output_path 
  -g gencode.v27.primary_assembly.annotation.gtf 
  -f spanki.genome.fasta 
  -t sim_rpk_file -bp 100 -frag 200 -ends 2 -m errorfree

This conmood outputs some files in which junc_coverage.txt is what we need.
Example

  python Spanki-master/bin/spankisim_transcripts 
  -o ./fastq_file/sim_rpk1A 
  -g gencode.v27.primary_assembly.annotation.gtf 
  -f GRCh38.primary_assembly.genome.spanki.fa 
  -t sim_rpk1A -bp 100 -frag 200 -ends 2 -m errorfree

Step1
perl sim_rpk.pl output_file_prefix gene_rpk degree_diff
Example
perl sim_rpk.pl sim_rpk1 50 8

Step2 generate a dice format file as input in step3
awk '{print $2}' rim_rpk_file |paste - 00.basic_infor/dice_order_four_col >sim_rpk_dice_file
Example
awk '{print $2}' sim_rpk1A |paste - 00.basic_infor/dice_order_four_col >sim_rpk1A_dice

Step3 simulate dropout event

  python simuDropout_modify.py 
  -a gencode.v27.primary_assembly.annotation.gtf 
  -f GRCh38.primary_assembly.genome.spanki.fa 
  -d sim_rpk_dice_file
  --dropoutRate 0.1 
  -o sim_rpk_dice_output_directory  
  -m errorfree

NOTE: This step will output error message.However, the dropout simulation that we exactly need has been outputed.
Example

  python simuDropout_modify.py 
  -a gencode.v27.primary_assembly.annotation.gtf 
  -f GRCh38.primary_assembly.genome.spanki.fa 
  -d sim_rpk1A_dice
  --dropoutRate 0.1 
  -o sim_rpk1A_dropout  
  -m errorfree

Step4

  python Spanki-master/bin/spankisim_transcripts 
  -o output_directory
  -g gencode.v27.primary_assembly.annotation.gtf 
  -f spanki.genome.fasta 
  -t dropout_tran_rpk/tran_rpk.txt -bp 100 -frag 200 -ends 2 -m errorfree

Example

  python Spanki-master/bin/spankisim_transcripts 
  -o ./fastq_file/sim_rpk1A 
  -g gencode.v27.primary_assembly.annotation.gtf 
  -f GRCh38.primary_assembly.genome.spanki.fa 
  -t sim_rpk1A/tran_rpk.txt -bp 100 -frag 200 -ends 2 -m errorfree