dndsR

Pre-release. Still performing tests before publication. Use at your own risk.

A scalable dN/dS analysis R package. For single or multiple pairwise comparisons of genomes, dndsR...

• Extracts CDS or proteins using input genome FASTAs and gene annotation GFF3s
• Calculates dN/dS
• Visualizes selection pressures across the genomes through an ideogram
• Calculates enrichment of gene functional annotation terms (IPR, GO, etc.) for biological functions under positive/diversifying selection
• Analyzes specified retgions for overall differences in dN/dS (i.e., do Dkmer regions of B and C have dN/dS>1 significantly more frequently than

dndsR is built for both containerized command line usage and as a loadable library in R.

Setup

Note: We currently recommend Docker and Singularity with CLI usage, however, if Orthofinder 2.5.4 and Diamond 2.1.14 are present on your system and R dependencies are installed, you can use the library directly in R. See Dockerfile for dependencies.

Clone the Repo

git clone https://github.com/Scrumpis/dndsR
chmod 774 -R dndsR

Pull the Image

Pull image from DockerHub

Singularity

singularity pull dndsr.sif docker://scrumpis/dndsr:latest

Docker

docker pull scrumpis/dndsr:latest

CLI Usage

Recommended for large-scale analysis. There is also an R.md vignette for users who want to work in Rstudio or similar. All commands allow single or batch comparisons. All functions will produce outputs for both the query and subject of a comparison by default.

Sample comparison_file

dndsR was built to run batches of comparisons and can optionally take as input a space or tab separated text file (comparison_file) containing: comparison_basename, "query_fasta", "query_gff3", "subject_fasta", "subject_gff3". Example:

Cform_v_Calbum "/path_to/Cformosanum.fasta" "/path_to/Cformosanum.gff" "/path_to/Calbum.fasta" "/path_to/Calbum.gff"
CalbumB_v_CalbumC "/path_to/CalbumB.fasta" "/path_to/CalbumB.gff" "/path_to/CalbumC.fasta" "/path_to/CalbumC.gff"

1. split_comparisons.R (optional)

Separate subgenomes, haplotypes, or other patterns into their own fastas and gffs and generates a new comparison_file corresponding to the splits. This is generally recommended for calculations between polyploid species so best matches occur between the same subgenome. If needed, SubPhaser can be used to phase allopolyploids lacking diploid progenitor genomes.

singularity exec dndsr.sif ./dndsR-launcher run split_comparisons \
-C comparison_file.txt -v -m subgenome

2. Extract CDS or Proteins

Extracts CDS or proteins into a new fasta using the genome.fasta and genome.gff files for each species of each comparison in comparison_file.

singularity exec dndsr.sif ./dndsR-launcher run extract_cds \
-C comparison_file.txt

3. Calculate dN/dS

Long runtime. If on cluster, consider submitting through SLURM, PBS, or similar.

singularity exec dndsr.sif ./dndsR-launcher run calculate_dnds \
-C comparison_file.txt -t 80

4. Append annotations

Appends GFF annotation attributes, functional terms, seqname, start, and end values for both query and subject to dN/dS calculations based on gene_id.

singularity exec dndsr.sif ./dndsR-launcher run append_annotations \
-C comparison_file.txt -O . -v -t 8

5. Annotation term enrichment

Enrichment of IPR terms under positive selection. Comparable to topGO in function. Handles parent-child relationships of IPR terms.

ipr_enrichment

singularity exec dndsr.sif ./dndsR-launcher run ipr_enrichment \
-C comparison_file.txt -t 8 -v -O .

go_enrichment

TopGO enrichment of GO terms under positive selection.

singularity exec dndsr.sif ./dndsR-launcher run go_enrichment \
-C comparison_file.txt -t 8 -v -O .

term_enrichment

General term enrichment. Fisher's Exact Test and multiple testing correction. Looks for non-IPR or GO terms like KEGG, PANTHER, etc. Optionally takes as input a custom pattern of interest to test for enrichment.

singularity exec dndsr.sif ./dndsR-launcher run term_enrichment \
-C comparison_file.txt -t 8 -v -O .

6. Selection pressure ideogram

Visualizes dN/dS binned values accross a genome in an ideogram.

singularity exec dndsr.sif ./dndsR-launcher run dnds_ideogram \
-C comparison_file.txt -t 8 -v -O .

7. Comparative Analysis

The below is for making comparisons between dN/dS outputs. Outputs dN/dS distributions and stats between comparisons. Requires regions.bed of interest for analysis (will update to default to regionless, whole genome analysis with regional option). regions.bed (seq_name, start, end, feature_name (optional)
Example:

Chr01B 71000000 72000000 SG3
Chr01B 72000000 73000000 SG3
Chr01B 73000000 74000000 SG3

Run regional_dnds_summary

singularity exec dndsr.sif ./dndsR-launcher run regional_dnds_summary \
-C comparison_file.txt \
--regions-bed regions.bed \
-O .

Contrast dndsR calculations (i.e., enrichment of regional selection pressures)

contrast_file.txt (new_comparison_name, query_comparison_name, query_genome, subject_comparison, subject_genome).
The query_ and subject_genome entries are the side of the comparison (query/subject) they belonged to in the referenced comparison. B was query in CalbumBvC and I want to use its dN/dS calculations and gene_ids, so I selected query, where subject would've used C's values.
Example:

BCvsBD CalbumBvC query CalbumUkBvD query
BCvsCD CalbumBvC subject CalbumUkCvD query

Run regional_dnds_contrasts

singularity exec dndsr.sif ./dndsR-launcher run regional_dnds_contrasts \
-C comparison_file.txt \
--regions-bed regions.bed \
--contrast-file contrast_file.txt \
-O .

Notes

Can use special characters or spaces in path if quoted in the comparison_file

Future Improvements

• ggplot::aes_string deprecated
• Update Rvignette with test dataset
• Cleanup documentation
• DBUS warning is container related, can be ignored, and will be updated
• Add forest plot function and other grand analyses
• Verify these are unusused and rebuild image: Biogenerics, IRanges, S4Vectors, optparse, readr
• Change regional analysis name. Can focus regions, but can do whole genome summaries too.
  • Change default to regions_bed = NULL
• Check if below is needed anymore

Install dndsR library

Singularity

singularity exec dndsr.sif ./dndsR-launcher install