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Project Objective:

Find the orthologs among the 5 species of beetles and annotate the orthologs.

Project Workflow:

  • Step 1: Construct a de novo transcriptome assembly.
    a. Run Trinity to construct a primary assembly.
    b. Run BUSCO to check the quality of the assembly. Use BUSCO.

  • Step 2: Identify the coding regions.
    a. Using generated transcriptome from the previous step, run LongOrfs with threshold set to at least 100 aa length for each ORF. Use TransDecoder.LongOrfs
    b. Using the predicted peptide sequences (.pep file) run BLASTP against Tribolium castaneum protein sequences. Use makeblastdb and blastp with -max_target_seqs 1 -outfmt 6 -evalue 1e-10 flags.

      # Get the sequence file.
  wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/002/335/GCF_000002335.3_Tcas5.2/GCF_000002335.3_Tcas5.2_protein.faa.gz
  # Uncompress the file.
  gzip -d GCF_000002335.3_Tcas5.2_protein.faa.gz
  # Make sure to create blastdb before this command.
  # Example blastp.
  blastp -query transdecoder_dir/longest_orfs.pep -db Tcas_protein_db.fasta  -max_target_seqs 1 -outfmt 6 -evalue 1e-10 -num_threads 48 > blastp.outfmt6

    c. Using the homology information from BLASTP, predict the coding sequence.

      TransDecoder.Predict -t target_transcripts.fasta --retain_blastp_hits blastp.outfmt6

    d. Run BUSCO to check the quality of the filtered transcriptome. Use BUSCO.

  • Step 3: Find the Orthologs among 5 species.
    a. Run all vs all BLAST among 5 species. Use makeblastdb and blastp. 
      wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/002/335/GCF_000002335.3_Tcas5.2/GCF_000002335.3_Tcas5.2_rna.fna.gz
  # Uncompress the file.
  gzip -d GCF_000002335.3_Tcas5.2_rna.fna.gz
  # Make sure to create blastdb before this command.
  # Example blastp.
  blastp -query predicted_peptides.pep -db Tcas_protein_db.fasta  -max_target_seqs 1 -outfmt 6 -num_threads 48 > species1_Tcas_db.txt

    b. Pick the reciprocal Best BLAST hit (RBBH). Write a python script.
    c. Run a 5 way script to pull out the orthologs among 5 species. Write a python script.

  • Step 4: Add annotation to the orthologs. a.Using T_cas ids and T_cas reference gff files, add chromosome id (for example: LGX, LG2 etc.),chromosome name (Autosome/ Sex chromosome). Write python script. 
      # Get the gff file.
  wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/002/335/GCF_000002335.3_Tcas5.2/GCF_000002335.3_Tcas5.2_genomic.gff.gz
  # Uncompress the file.
  gzip -d GCF_000002335.3_Tcas5.2_genomic.gff.gz

Helpful Hints:

  • To use BUSCO, the first step is to get the lineage and then busco can be run as follows.

      # Get the lineage
  wget https://busco.ezlab.org/datasets/endopterygota_odb9.tar.gz
  # Uncompress the directory.
  tar xvf endopterygota_odb9.tar.gz --gunzip
  # Activate busco environment
  conda activate busco
  # Run BUSCO
  run_busco --in transcriptome.fasta --out [output_directory_name] -l [path_to_]endopterygota_odb9 -m tran -c 48

  • TransDecoder has two programs. These two programs can be run as follows.

      # To run LongOrfs with minimum ORF length as 100 amino acids.
  TransDecoder.LongOrfs -t target_transcripts.fasta -m 100
  # To run Predict
  TransDecoder.Predict -t target_transcripts.fasta --retain_blastp_hits blastp.outfmt6

Reference:

High Quality de Novo Transcriptome Assembly of Croton tiglium. - PDF