This tutorial uses the test data assembly.fasta, a small set of de novo transcriptome contigs, located in the test sub-directory of PlantTribes installation to show how to perform an analysis using the various pipelines of PlantTribes.
1). The following command will post processes assembly.fasta using ESTScan coding regions prediction method with aid of Arabidopsis thaliana references matrices in strand specific mode, and removes similar (sub)sequences and sequences shorter than 200 bp.
PlantTribes/pipelines/AssemblyPostProcessor --transcripts assembly.fasta --prediction_method estscan --score_matrices /path/to/score/matrices//Arabidopsis_thaliana.smat --strand_specific --dereplicate --min_length 200
2). The following command as in 1) above will post processes assembly.fasta using TransDecoder coding regions prediction method in strand specific mode, and remove similar (sub)sequences and sequences shorter than 200 bp.
PlantTribes/pipelines/AssemblyPostProcessor --transcripts assembly.fasta --prediction_method transdecoder --strand_specific --dereplicate --min_length 200
Output:
assemblyPostProcessing_dir/transcripts.cds
assemblyPostProcessing_dir/transcripts.pep
assemblyPostProcessing_dir/transcripts.cleaned.cds
assemblyPostProcessing_dir/transcripts.cleaned.cds
assemblyPostProcessing_dir/transcripts.cleaned.nr.cds
assemblyPostProcessing_dir/transcripts.cleaned.nr.pep
3). Including the targeted gene family options to the commands in 1) and 2) attempt to reassemble fragmented contigs assigned to targeted gene families (orthogroups) listed in the targetOrthos.ids file located in the test sub-directory of PlantTribes installation into contiguous transcripts whenever possible.
PlantTribes/pipelines/AssemblyPostProcessor --transcripts assembly.fasta --prediction_method transdecoder --gene_family_search targetOrthos.ids --scaffold 22Gv1.1 --method orthomcl --strand_specific --dereplicate --min_length 200 --num_threads 10
Output:
assemblyPostProcessing_dir/targeted_gene_family_assemblies/213.fasta
assemblyPostProcessing_dir/targeted_gene_family_assemblies/213.fna
assemblyPostProcessing_dir/targeted_gene_family_assemblies/213.faa
assemblyPostProcessing_dir/targeted_gene_family_assemblies.stats
1). Gene family classification of the post processed assembly.fasta de novo transcripts using BLASTP as a classifier - faster.
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier blastp --num_threads 10
Output:
geneFamilyClassification_dir/proteins.blastp.22Gv1.1
geneFamilyClassification_dir/proteins.blastp.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.blastp.22Gv1.1.bestOrthos.summary
2). Gene family classification of the post processed assembly.fasta de novo transcripts using HMMScan as a classifier - slower but more sensitive to remote homologs.
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier hmmscan --num_threads 10
Output:
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1.bestOrthos.summary
3). Gene family classification of the post processed assembly.fasta de novo transcripts using both BLASTP and HMMScan as a classifiers - more exhaustive.
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier both --num_threads 10
Output:
geneFamilyClassification_dir/proteins.blastp.22Gv1.1
geneFamilyClassification_dir/proteins.blastp.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos.summary
4). Customizing the selection of single/low copy gene families by taxa. Example of a customized single/low copy selection configuration file, 22Gv1.1.singleCopy.config is located in the config sub-directory of PlantTribes installation.
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier both --single_copy_custom --num_threads 10
5). Alternative single/low copy gene families selection command
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier both --single_copy_taxa 20 --taxa_present 21 --num_threads 10
Output:
geneFamilyClassification_dir/proteins.blastp.22Gv1.1
geneFamilyClassification_dir/proteins.blastp.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos.summary
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos.summary.singleCopy
6). Creating gene family fasta files (cds and their corresponding peptide) for the post processed de novo transcriptome assembly.
PlantTribes/pipelines/GeneFamilyClassifier --proteins assemblyPostProcessing_dir/transcripts.cleaned.nr.pep --scaffold 22Gv1.1 --method orthomcl --classifier both --single_copy_taxa 20 --taxa_present 21 --num_threads 10 --orthogroup_fasta --coding_sequences assemblyPostProcessing_dir/transcripts.cleaned.nr.cds
Output:
geneFamilyClassification_dir/proteins.blastp.22Gv1.1
geneFamilyClassification_dir/proteins.blastp.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1
geneFamilyClassification_dir/proteins.hmmscan.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos.summary
geneFamilyClassification_dir/proteins.both.22Gv1.1.bestOrthos.summary.singleCopy
geneFamilyClassification_dir/orthogroups_fasta - transcriptome assembly orthogroup fasta directory
geneFamilyClassification_dir/single_copy_fasta - transcriptome assembly single/low copy orthogroup fasta directory
1). Integrating classified post processed de novo transcriptome assembly sequence(s) with the scaffold gene family sequences
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --orthogroup_fna
Output:
phylogenomicsAnalysis_dir/orthogroups_fasta/ - orthogroup fasta directory
2). Creating gene family multiple sequence alignments using MAFFT L-INS-i iterative refinement method
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --create_alignments
3). Adding unaligned post processed de novo transcriptome assembly sequence(s) into precomputed scaffold gene family multiple sequence alignments - faster
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --add_alignments
4). Creating gene family multiple sequence alignments using PASTA (Practical Alignment using SATe and Transitivity) method - for larger data sets
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --pasta_alignments
Output:
phylogenomicsAnalysis_dir/orthogroups_fasta/ - orthogroup fasta directory
phylogenomicsAnalysis_dir/orthogroups_aln/ - orthogroup multiple sequence alignments directory
5). Building maximum-likelihood gene family phylogenetic trees with RAxML
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --pasta_alignments --tree_inference raxml
6). Building approximately-maximum-likelihood gene family phylogenetic trees with FastTree - faster
PlantTribes/legacy/PhylogenomicsAnalysis --orthogroup_faa geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl --pasta_alignments --tree_inference fasttree
Output:
phylogenomicsAnalysis_dir/orthogroups_fasta/ - orthogroup fasta directory
phylogenomicsAnalysis_dir/orthogroups_aln/ - orthogroup multiple sequence alignments directory
phylogenomicsAnalysis_dir/orthogroups_tree/ - orthogroup phylogenetic trees directory
1). Integrating classified post processed de novo transcriptome assembly sequence(s) with the scaffold gene family sequences
PlantTribes/pipelines/GeneFamilyIntegrator --orthogroup_fasta geneFamilyClassification_dir/orthogroups_fasta --scaffold 22Gv1.1 --method orthomcl
Output:
integratedGeneFamilies_dir/ - orthogroup fasta directory
1). Creating gene family multiple sequence alignments using MAFFT L-INS-i iterative refinement method
PlantTribes/pipelines/GeneFamilyAligner --orthogroup_faa integratedGeneFamilies_dir --alignment_method mafft
2). Creating gene family multiple sequence alignments using PASTA (Practical Alignment using SATe and Transitivity) method - for larger data sets
PlantTribes/pipelines/GeneFamilyAligner --orthogroup_faa integratedGeneFamilies_dir --alignment_method pasta --pasta_script_path /path/to/pasta-code/pasta/run_pasta.py
Output:
geneFamilyAlignments_dir/orthogroups_aln_faa - orthogroup multiple sequence alignments directory
1). Building maximum-likelihood gene family phylogenetic trees with RAxML
PlantTribes/pipelines/GeneFamilyPhylogenyBuilder --orthogroup_aln geneFamilyAlignments_dir/orthogroups_aln_faa --scaffold 22Gv1.1 --method orthomcl --tree_inference raxml
Output:
geneFamilyPhylogenies_dir/phylip_aln/ - orthogroup phylip multiple sequence alignments directory
geneFamilyPhylogenies_dir/orthogroups_tree/ - orthogroup phylogenetic trees directory
2). Building approximately-maximum-likelihood gene family phylogenetic trees with FastTree - faster
PlantTribes/pipelines/GeneFamilyPhylogenyBuilder --orthogroup_aln geneFamilyAlignments_dir/orthogroups_aln_faa --tree_inference fasttree
Output:
geneFamilyPhylogenies_dir/orthogroups_tree/ - orthogroup phylogenetic trees directory
1). Performing paralogous ks analysis limiting ks values between 0.02 and 4.0
PlantTribes/pipelines/KaKsAnalysis --coding_sequences_species_1 PlantTribes/test/species1.fna --proteins_species_1 PlantTribes/test/species1.faa --comparison paralogs --min_ks 0.02 --max_ks 4.0 --num_threads 10
output:
kaksAnalysis_dir/species1.fna - species1 input coding sequences (CDS)
kaksAnalysis_dir/species1.faa - species1 input amino acids (proteins)
kaksAnalysis_dir/species1.fna.blastn.paralogs - species1 self blastn results
kaksAnalysis_dir/species1.fna.blastn.paralogs.rbhb - species1 paralogous pairs
kaksAnalysis_dir/species1.fna.blastn.paralogs.rbhb.kaks - species1 ka/ks analysis results
2). Performing orthologous ks analysis limiting ks values between 0.02 and 4.0
PlantTribes/pipelines/KaKsAnalysis --coding_sequences_species_1 PlantTribes/test/species1.fna --proteins_species_1 PlantTribes/test/species1.faa --comparison orthologs --coding_sequences_species_2 PlantTribes/test/species2.fna --proteins_species_2 PlantTribes/test/species2.faa --min_ks 0.02 --max_ks 4.0 --num_threads 10
output:
kaksAnalysis_dir/species1.fna - species1 input coding sequences (CDS)
kaksAnalysis_dir/species1.faa - species1 input amino acids (proteins)
kaksAnalysis_dir/species2.fna - species2 input coding sequences (CDS)
kaksAnalysis_dir/species2.faa - species2 input amino acids (proteins)
kaksAnalysis_dir/species1.fna.blastn.orthologs - species1 vs species2.blastdb blastn results
kaksAnalysis_dir/species2.fna.blastn.orthologs - species2 vs species1.blastdb blastn results
kaksAnalysis_dir/species1and2.fna.blastn.orthologs.rbhb - species1 vs species2 orthologous pairs
kaksAnalysis_dir/species1and2.fna.blastn.orthologs.rbhb.kaks - species1 vs species2 ka/ks analysis results
3). Performing paralogous ks analysis limiting ks values between 0.02 and 4.0, and fitting upto 4 mixture model of multivariate normal components to identify significant duplication event(s) in a genome
PlantTribes/pipelines/KaKsAnalysis --coding_sequences_species_1 PlantTribes/test/species1.fna --proteins_species_1 PlantTribes/test/species1.faa --comparison paralogs --fit_components --num_of_components 4 --min_ks 0.02 --max_ks 4.0 --num_threads 10
output:
kaksAnalysis_dir/species1.fna - species1 input coding sequences (CDS)
kaksAnalysis_dir/species1.faa - species1 input amino acids (proteins)
kaksAnalysis_dir/species1.fna.blastn.paralogs - species1 self blastn results
kaksAnalysis_dir/species1.fna.blastn.paralogs.rbhb - species1 paralogous pairs
kaksAnalysis_dir/species1.fna.blastn.paralogs.rbhb.kaks - species1 ka/ks analysis results
kaksAnalysis_dir/species1.fna.blastn.paralogs.rbhb.kaks.components - significant components in the ks distribution of species1
4). Performing orthologous ks analysis limiting ks values between 0.02 and 4.0, and fitting upto 4 mixture model of multivariate normal components to identify significant duplication event(s) in a genome
PlantTribes/pipelines/KaKsAnalysis --coding_sequences_species_1 PlantTribes/test/species1.fna --proteins_species_1 PlantTribes/test/species1.faa --comparison orthologs --coding_sequences_species_2 PlantTribes/test/species2.fna --proteins_species_2 PlantTribes/test/species2.faa --fit_components --num_of_components 4 --min_ks 0.02 --max_ks 4.0 --num_threads 10
output:
kaksAnalysis_dir/species1.fna - species1 input coding sequences (CDS)
kaksAnalysis_dir/species1.faa - species1 input amino acids (proteins)
kaksAnalysis_dir/species2.fna - species2 input coding sequences (CDS)
kaksAnalysis_dir/species2.faa - species2 input amino acids (proteins)
kaksAnalysis_dir/species1.fna.blastn.orthologs - species1 vs species2.blastdb blastn results
kaksAnalysis_dir/species2.fna.blastn.orthologs - species2 vs species1.blastdb blastn results
kaksAnalysis_dir/species1and2.fna.blastn.orthologs.rbhb - species1 vs species2 orthologous pairs
kaksAnalysis_dir/species1and2.fna.blastn.orthologs.rbhb.kaks - species1 vs species2 ka/ks analysis results
kaksAnalysis_dir/species1and2.fna.blastn.orthologs.rbhb.kaks.components - significant components in the ks distribution of species1 vs species2
Consult the PlantTribes manual for usage of other optimization options not used in this tutorial.