WHI

A genome-based method embracing evolutionary distance and ecology to delineate prokaryotic species

Test on NCBI genomes

1. Genome collection

1.1 Download assembly_summary.txt

For archea genomes:
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/archea/assembly_summary.txt
mv assembly_summary.txt Archea_assembly_summary.txt
Note: The data used in our paper is Data/Archea_assembly_summary.txt. You should use this data to repeat our study. Please note that if you download assembly_summary.txt by yourself, the results may be slightly different because of updated NCBI database.

For eubacteria genomes:
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/bacteria/assembly_summary.txt
mv assembly_summary.txt Bacteria_assembly_summary.txt
Note: The data used in our paper is Data/Archea_assembly_summary.txt. You should use this data to repeat our study. Please note that if you download assembly_summary.txt by yourself, the results may be slightly different because of updated NCBI database.

Merging two files:
cat Archea_assembly_summary.txt Bacteria_assembly_summary.txt >Total_assembly_summary.txt

1.2. Generate script for downloading genomes

perl bin/01.GenerateShell_4wget.pl Total_assembly_summary.txt Genome wget.sh

1.3. Download and decompress genomes

sh wget.sh

Decompress files:
perl bin/02.GenerateShell_4Gzip.pl Genome gzip.sh
sh gzip.sh

1.4 Generate genome list

To generate a table containing assembly_accession,species_taxid,organism_name,infraspecific_name, assembly_level,and file path:
perl bin/03.Generate_GenomeList.pl Total_assembly_summary.txt Genome Total_Genome.list Total_Genome.log

1.5. Separate Chromosomes and Plasmids

To separate chromosomes and plasmids, and then calculate the genome sizes for chromosomes:
perl bin/04.Separate_Chrom_Plasmid.pl Total_Genome.list data/Plasmids.ids Total_Chrom_Plasmid.list Chromosome_Plasmid Plasmids.ids: downloaded from NCBI(wget ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_refseq/Plasmids/Plasmids.ids)

2. Validated Species and type strains

Species with validated names were collected from the List of Prokaryotic names with Standing in Nomenclature (LPSN) database (http://www.bacterio.net/), which are included in Data/Validated_SpeciesName.xls.

Type strains were recognized using the Straininfo bioportal (http://www.straininfo.net/) and LPSN. Collected type strains are included in Data/Type_strain.xls.

3. Genome selection

3.1 Select genomes with validated species names

To discard genomes without validated species name and retain genomes belonging to validated species
perl bin/05.ValidatedSpecies_GenomeInfo.pl data/Validated_SpeciesName.xls Total_Chrom_Plasmid.list ValidatedSpecies_GenomeInfo.xls

3.2 Select draft genomes with size >0.5 megabase-pairs (Mb)

To filter out low-coverage genomes (< 0.5 Mb):
perl bin/06.ValidatedGenome_more500kb.pl ValidatedSpecies_GenomeInfo.xls ValidatedGenome_more500kb.xls

4. Reference and query genomes

To select reference and query genomes:
perl bin/07.Ref_Query_GenomeInfo.pl ValidatedGenome_more500kb.xls data/Type_strain.xls Ref_GenomeInfo.xls Query_GenomeInfo.xls

5. Sieved by TETRA

5.1 Calculating zvalues

For ref. genomes:
perl bin /08.Zvalue.pl Ref_GenomeInfo.xls Ref_Zvalue.xls
For Query genomes:
perl bin /08.Zvalue.pl Query_GenomeInfo.xls Query_Zvalue.xls

5.2 Calculating TETRAs and selecting pairs for WHI and ANI calculation

perl 09.Pairs_byTETRA.pl Ref_Zvalue.xls Query_Zvalue.xls Pairs_byTETRA.xls

6. Calculation of ANI, PSG and WHI

6.1 Generate script for alignments

perl bin/10.GenerateShell_4GenomeAlign.pl Ref_GenomeInfo.xls Query_GenomeInfo.xls Pairs_byTETRA.xls GenomeAlign GenomeAlign.sh Delta.list
sh GenomeAlign.sh

6.2 Calculation of ANI, PSG and WHI

cut -f 1,6 ValidatedGenome_more500kb.xls >Genome_size.xls
perl bin/11.ANI_WHI_PSG.pl Delta.list Genome_Size.xls ANI_WHI_PSG.xls

7. Species delineation using the Best Match strategy

7.1 Species delineation by WHI

perl bin/12.SpeciesDelineation_byWHI_BestMatch.pl ANI_WHI_PSG.xls SpeciesDelineation_byWHI_BestMatch.xls

7.2 Species delineation by ANI

perl bin/12.SpeciesDelineation_byANI_BestMatch.pl ANI_WHI_PSG.xls SpeciesDelineation_byANI_BestMatch.xls

8. Validation by NCBI taxonomy

8.1 Validate delineation by WHI

perl bin/13.Validate_4WHI_BestMatch.pl SpeciesDelineation_byWHI_BestMatch.xls Ref_GenomeInfo.xls Query_GenomeInfo.xls WHI_validate_detail.xls WHI_validate_stat.xls
Then you can use bin/DrawFigS6_Rcode.R to draw a figure (Fig. S6B)

8.2 Validate delineation by ANI

perl bin/13.Validate_4ANI_BestMatch.pl SpeciesDelineation_byANI_BestMatch.xls Ref_GenomeInfo.xls Query_GenomeInfo.xls ANI_validate_detail.xls ANI_validate_stat.xls  

8.3 Recall, Precision and F-score

perl bin/14.Fscore.pl WHI_validate_detail.xls ANI_validate_detail.xls 61914 Recall_Precision_Fscore.xls

9. Species delineation using the Greedy-Match strategy

9.1 Validate delineation by WHI

perl bin/15.Validate_4WHI_GreedyMatch.pl ANI_WHI_PSG.xls Ref_GenomeInfo.xls Query_GenomeInfo.xls Validate_4WHI_GreedyMatch_detail.xls Validate_4WHI_GreedyMatch_stat.xls

9.2 Validate delineation by ANI

perl bin/15.Validate_4ANI_GreedyMatch.pl ANI_WHI_PSG.xls Ref_GenomeInfo.xls Query_GenomeInfo.xls Validate_4ANI_GreedyMatch_detail.xls Validate_4ANI_GreedyMatch_stat.xls

Then you can use bin/14.Fscore.pl to calculate recall, precision and F-score.

Test on DDH database

The extended DDH database: data/DDH_Database.xls
Following the below procedures, you will obtains the results for DDH database: data/DDH_Results.xls

1. Gain genome information

perl bin/16.DDH_GenomeInfo.pl Total_Chrom_Plasmid.list data/DDH_Database.xls DDH_GenomeInfo.xls Total_Chrom_Plasmid.list: to get this file, you should follow the section "Test on NCBI genomes (step 1.1 to 1.5)"

2. Calculate Z-values for DDH genomes

perl bin/08.Zvalue.pl DDH_GenomeInfo.xls DDH_Zvalue.xls

3. Calculated TETRAs

perl bin/17.DDH_TETRA.pl DDH_Zvalue.xls DDH_Database.xls DDH_TETRA.xls

4. Generate script for alignments

perl bin/18.GenerateShell_4DDHGenomeAlign.pl DDH_GenomeInfo.xls data/DDH_Database.xls DDH_GenomeAlign DDH_GenomeAlign.sh DDH_Delta.list sh DDH_GenomeAlign.sh

5. Calculation of ANI, PSG and WHI

cut -f 1,6 DDH_GenomeInfo.xls >DDH_Genomesize.xls
perl bin/11.ANI_WHI_PSG.pl DDH_Delta.list DDH_Genomesize.xls DDH_ANI_WHI_PSG.xls

6. Generate final results

perl bin/19.DDH_Results.pl data/DDH_Database.xls DDH_ANI_WHI_PSG.xls DDH_TETRA.xls DDH_Results.xls

Please Cite

If you use WHI in your publication, please cite:

Support

If you are having issues, please email me via [email protected]