Interpreting the Results¶

EnTAP provides many output files at each stage of execution to better see how the data is being managed throughout the pipeline:

Expression Filtering
Frame Selection
Similarity Searching
Orthologous Groups/Ontology
Final Annotation Results

Expression Filtering¶

The /expression folder will contain all of the relevant information for this stage of the pipeline.

Frame Selection (GeneMarkS-T)¶

The /frame_selection folder will contain all of the relevant information for the frame selection stage of the pipeline. This folder will contain the main files (results from frame selection software), files processed from EnTAP, and figures generated from EnTAP.

GeneMarkS-T Files: /frame_selection¶

The files within the root /frame_selection directory contain the results from the frame selection portion of the pipeline. More information can be found at GeneMarkS-T. With a generic transcriptome input of “Species.fasta”, these files will have the following format:

Species.fasta.fnn
- Nucleotide fasta formatted frame selected sequences
Species.fasta.faa
- Amino acid fasta formatted frame selected sequences
Species.fasta.lst
- Information on each sequence (partial/internal/complete/ORF length)
.err and .out file
- These files are will contain any error or general information produced from the GeneMarkS-T run

EnTAP Files: /processed¶

Files within the /processed are generated by EnTAP and will contain ORF information based on the GeneMarkS-T execution.

complete_genes.fasta
- Amino acid sequences of complete genes from transcriptome
partial_genes.fasta
- Amino acid sequences of partial (5’ and 3’) sequences
internal_genes.fasta
- Amino acid sequences of internal sequences
sequences_lost.fasta
- Nucleotide sequences in which a frame was not found. These will not continue to the next stages of the pipeline

EnTAP Files: /figures¶

In addition to files, EnTAP will generate figures within the /figures directory. These are some useful visualizations of the information provided by GeneMarkS-T

frame_results_pie.png
- Pie chart representing the transcriptome (post expression filtering) showing complete/internal/partial/and sequences in which a frame was not found
frame_selected_seq.png
- Box plot of sequence length vs. the sequences that were lost during frame selection and the sequences in which a frame was found

Similarity Search (DIAMOND)¶

The /similarity_search directory will contain all of the relevant information for the similarity searching stage of the pipeline. This folder will contain the main files (results from similarity search software), files analyzing hits from each database, overall results combining the information from each database, and figures generated from EnTAP.

DIAMOND Files: /similarity_search¶

The files within the /similarity_search directory contain the results from the similarity searching portion of the pipeline against each database you select. More information can be found at DIAMOND. With running blastp (protein similarity searching), a generic transcriptome input of “Species.fasta”, with a database called “database” the files will have the following format:

blastp_Species_database.out
- This contains the similarity search information provided in the format from DIAMOND
- Header information (from left to right):
  Query Sequence ID
  
  Subject Sequence ID
  
  Percentage of Identical Matches
  
  Alignment Length
  
  Number of Mismatches
  
  Number of gap openings
  
  Start of alignment in query
  
  End of alignment in query
  
  Start of alignment in subject
  
  End of alignment in subject
  
  Expect (e) value
  
  Bit score
  
  Query Coverage
  
  Subject Title (pulled from database)
blastp_Species_database_std.err and .out
- These files are will contain any error or general information produced from DIAMOND

EnTAP Files: /processed¶

Files within the /processed are generated by EnTAP and will contain information based on the hits returned from similarity searching against each database. This information contains the best hits (discussed previously) from each database based on e-value, coverage, informativeness, phylogenetic closeness, and contaminant status.

The files below represent a run with the same parameters as the section above:

All the TSV files mentioned in this section will have the same header as follows (from left to right):
- Query sequence ID
- Subject sequence ID
- Percentage of identical matches
- Alignment length
- Number of mismatches
- Number of gap openings
- Start of alignment in query
- End of alignment in query
- Start of alignment in subject
- End of alignment in subject
- Expect (e) value
- Query coverage
- Subject title
- Species (pulled from hit)
- Origin Database
- ORF (taken from frame selection stage)
- Contaminant (yes/no the hit was flagged as a contaminant)
database/best_hits.faa and .fnn and .tsv
- Best hits (protein and nucleotide) that were selected from this database
- This contains ALL best hits, including any contaminants that were found as well as uninformative hits
- The .tsv file contains the header information mentioned above of these same sequences
- Note: Protein or nucleotide information may not be available to report depending on your type of run (these files will be empty)
database/best_hits_contam.faa/.fnn/.tsv
- Contaminants (protein/nucleotide) separated from the best hits file. As such, these contaminants will also be in the _best_hits.faa/.fnn.tsv files
database/best_hits_no_contam.faa/.fnn/.tsv
- Sequences (protein/nucleotide) that were selected as best hits and not flagged as contaminants
- With this in mind: best_hits = best_hits_no_contam + best_hits_contam
- These sequences are separated from the rest for convenience if you would like to examine them differently
database/no_hits.faa/.fnn/.tsv
- Sequences (protein/nucleotide) from the transcriptome that did not hit against this particular database.
- This does not include sequences that were lost during expression filtering or frame selection
database/unselected.tsv
- Similarity searching can result in several hits for each query sequence. With only one best hit being selected, the rest are unselected and end up here
- Unselected hits can be due to a low e-value, coverage, or other properties EnTAP takes into account when selecting hits

EnTAP Files: /overall_results¶

While the /processed directory contains the best hit information from each database, the /overall_results directory contains the overall best hits combining the hits from each database.

EnTAP Files: /figures¶

In addition to files, EnTAP will generate figures within the /figures directory for each database. These are some useful visualizations of the information provided by similarity searching.

Here, there will be several figures:

species_bar.png / species_bar.txt
- Bar graph representing the top 10 species that were hit within a database
- Text file representing the data being displayed
contam_bar.png / contam_bar.txt
- Bar graph representing the top 10 contaminants (within best hits) that were hit against the databast
- Text file representing the data being displayed

Orthologous Groups/Ontology (EggNOG)¶

The /ontology directory will contain all of the relevant information for the EggNOG stage of the pipeline. This folder will contain the EggNOG files, files analyzing the annotation from EggNOG, and figures generated from EnTAP.

EggNOG Files: /ontology¶

Files within the /ontology are generated by EggNOG and will contain information based on the hits returned from EggNOG against the orthologous databases. More information can be found at EggNOG.

annotation_results.emapper.annotations
- EggNOG results for sequences that previously hit against DIAMOND databases in similarity searching
annotation_results_no_hits.emapper.annotations
- EggNOG results for sequences that previously did NOT hit against DIAMOND databases in similarity searching

EnTAP Files: /processed¶

EnTAP Files: /figures¶

Final Annotations¶

The final EnTAP annotations are contained within the /outfiles directory. These files are the summation of each stage of the pipeline and contain the combined information. So these can be considered the most important files!

All .tsv files in this section will have the following header information (from left to right)

Query sequence ID

Subject sequence ID

Percentage of identical matches

Alignment length

Number of mismatches

Number of gap openings

Start of alignment in query

End of alignment in query

Start of alignment in subject

End of alignment in subject

Expect (e) value

Query coverage

Subject title

Species (DIAMOND)

Origin Database (DIAMOND)

ORF (GeneMarkS-T)

Contaminant (yes/no the hit was flagged as a contaminant)

Seed ortholog (EggNOG)

Seed E-Value (EggNOG)

Seed Score (EggNOG)

Predicted Gene (EggNOG)

Taxonomic Scope (EggNOG, tax scope that was matched)

OGs (EggNOG, orthologous groups assigned)

Description (EggNOG)

KEGG Terms (EggNOG)

Protein Domains (EggNOG)

GO Biological (Gene Ontology normalized terms)

GO Cellular (Gene Ontology normalized terms)

GO Molecular (Gene Ontology normalized terms)

Gene ontology terms are normalized to levels based on the input flag from the user (or the default of 0,3,4). A level of 0 within the filename indicates that ALL GO terms will be printed to the annotation file. Normalization of GO terms to levels is generally done before enrichment analysis and is based upon the hierarchical setup of the Gene Ontology database. More information can be found at GO.

final_annotations_lvlX.tsv

As mentioned above, the ‘X’ represents the normalized GO terms for the annotation

This .tsv file will have the headers as mentioned previously as a summary of the entire pipeline

final_annotated.faa / .fnn

Nucleotide and protein fasta files containing all sequences that either hit databases through similarity searching or through the ontology stage

final_unannotated.aa / .fnn

Nucleotide and protein fasta files containing all sequences that did not hit either through similarity searching nor through the ontology stage