This repository includes code used in the article:

Phylogenomics and metabolic engineering reveal a conserved gene cluster in Solanaceae plants for withanolide biosynthesis

https://doi.org/10.1101/2024.09.27.614867

Building the Circos Plot:(Withanolide_biosynthesis/Circos)

1. extract_top_seq.py: Extract the top n longest sequences from an assembly fasta file.

   Usage:

    python3 extract_top_seq.py
   
    --in    <Full path to assembly fasta file>
    --out   <Full path to output text file>
    --n     <INT, number of chromosomes/contigs you want to show in the circos plot>
   

2. karyotype.py: Generate a Circos karyotype file from an assembly FASTA file.

   Usage:

    python3 karyotype.py
   
    --input,  -i  <Path to the input fasta file>
    --output, -o  <Path to the output karyotype file>
    --color,  -c  <Color for all chromosomes/contigs. Default is RdGy-3-div-3>
   

   If you want to change the chromosomes/contigs names on the circos plot, edit the 4th column in the output karyotype file accordingly. For circos-format color definitions, please see the documentation at https://circos.ca/documentation/tutorials/configuration/colors/.

3. gene_density.py: Calculates gene density per chromosome/contig from a gff3 file

   Usage:

   python3 gene_density.py <gff_file> <karyotype_file> <window_size>
   
   gff_file         Path to the GFF file containing gene information
   karyotype_file   Path to the karyotype file (Created in the previous step) 
   window_size      The size of the windows for gene density calculation (e.g., 1000000 for 1Mb windows)
   

4. tandem_repeat_density.py: Parses Tandem Repeat Finder TRF .dat output file and creates Circos format file

   Usage:

   python3 tandem_repeat_density.py <trf_outfile> <karyotype_file> <output_file> <bin_size>
   
   trf_outfile     Path to the TRF output file.
   karyotype_file  Path to the Circos karyotype file.
   output_file     Output file for Circos format.
   bin_size        Bin size
   -h, --help      Shows help message
   

5. TE_density.py: Calculates the percentage of TEs from gff3 output file of EDTA

   Usage:

   python3 TE_density.py <karyotype_file> <gff3_file> <so_numbers> <bin_size> <output_file>
   
   -h, --help          shows help message and exit
   karyotype_file      Path to the karyotype file.
   gff3_file           Path to the Whole-genome TE annotation ($genome.mod.EDTA.TEanno.gff3) file from EDTA.
   so_numbers          Comma-separated sequence ontology numbers (e.g., SO:0002280,SO:0002281).
   bin_size            Bin size 
   output_file         Output file to save the density information.
   

   Please refer to the TE sequence ontology file for the sequence ontology numbers. To display a single superfamily, use a single ontology number (e.g., SO:0002264 for Copia-type LTR retrotransposon) .To display an entire order, use multiple comma-separated sequence ontology numbers that constitute that order.

6. GC_density.py: Calculate GC content of input Fasta sequence in non-overlapping windows and write to an output file

   Usage:

    python3 GC_density.py -f input.fa [-b 1000] -o output.txt
   
    -h, --help                    shows help message and exit
    -f FASTA, --file=FASTA        Input Fasta format file
    -b BINSIZE, --bin=BINSIZE     default:1000 Bin size for non-overlapping windows
    -o OUTPUT, --output=OUTPUT    Output file to write the GC content
   

7. circos.conf: Circos configuration file used in this study

Building the Expression Heatmap: (Withanolide_biosynthesis/Heatmap)

Expression_hm.R: R code to plot the expression heatmap

Data availability

The raw sequencing data for Withania somnifera is available through the European Nucleotide Archive Project PRJEB64854. Root gene expression raw data is available at ERR13615536. The genome assembly and gene annotation sequences for W. somnifera are available via LeoPARD at https://leopard.tu-braunschweig.de/receive/dbbs_mods_00077979. Physalis pruinosa and P. grisea gene re-annotated sequences are available at https://leopard.tu-braunschweig.de/receive/dbbs_mods_00077980. Code used throughout the article is available at the GitHub repository: https://github.com/NancyChoudhary28/Withanolide_biosynthesis/

References

If you find the code useful in your research, please cite:

Hakim S.E.*, Choudhary N.*, Malhotra K.*, Peng J.*, Arafa A., Bültemeier A., Friedhoff R., Bauer M., Witte C.-P., Herde M., Heretsch P., Pucker B., Franke J. (2024). Phylogenomics and metabolic engineering reveal a conserved gene cluster in Solanaceae plants for withanolide biosynthesis. bioRxiv 2024.09.27.614867; doi: 10.1101/2024.09.27.614867.