LAST, Biopython, Samtools, UCSC Kent Utilities, ROAST  

   git clone https://github.com/liangpingping/CNSpipeline.git

   mv CNSpipeline /to/your/working/path/

 Step1: Prepare reference genome for subsequent alignment.

         python del-reference.py reference_genome.fa reference_genome_name

 Step2: Pairwise alignment and polish the alignment.

         python del-query.py query_genome.fa number_of_thread reference_genome_name

 Step3: cd to directory reference/tba and run Multiple pairwise.

         roast X=0 E=reference_genome_name species-guided-tree all_pairwise_alignment_file output_maf_file 

 Step4: Calculating the score of each base.

         python score-for-bases.py maf_file num_species
		 
 Step5: Give a cutoff value to get CNSs according to the coverage of CDS in reference genome.
 
         python get-cns.py score-file CDS-file cutoff