Plasmid Fusion Analysis Pipeline

Overview

This R script analyzes plasmid fusion events. It generates circular genome visualizations showing gene annotations and BLAST mapping results to identify potential plasmid fusion events.

Requirements

R Version

• R >= 4.3.0

Required R Packages

circlize
GenomicRanges
data.table
ggplot2
grid
IRanges
paletteer

Installation

install.packages(c("circlize", "data.table", "ggplot2", "grid", "paletteer"))
if (!require("BiocManager", quietly = TRUE))
  install.packages("BiocManager")
BiocManager::install(c("GenomicRanges", "IRanges"))

Input Files

Required Input Files

0. Genome file:

• plasmid_query.fa - Each plasmid recovered and contigs named with filename as the beginning as "filename_number"
• plasmid_collection.fa - The collection of all query plasmids

1. Plasmid annotation files:

• OMAP_pls.tsv - Processed plasmid data by OMAP-KP
• OMAP_pls_raw.tsv - Raw plasmid sequences by OMAP-KP

2. BLAST results:

• blast.out - BLAST alignment results

makeblastdb -in plasmid_collection.fa -dbtype nucl  -parse_seqids -out plasmid_collection_db

blastn -query plasmid_query.fa  -db plasmid_collection_db  -evalue 1e-10  -num_threads 10 -outfmt '6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qcovs qcovhsp slen qlen' -dust no -soft_masking false -max_target_seqs 100000000 | awk -v filename="$filename" '{print filename"\t"$0}' > blast.out

3. Annotation files:

• mob_type.tsv - MOB-typing results with replicon types and mobility predictions
• bakta_anno.tsv - Bakta gene annotations tsv file

mob_typer --infile  plasmid_query.fa  --out_file mob_type.tsv
bakta --output ./ --prefix plasmid_query   --meta  --keep-contig-headers  --locus-tag plasmid_query    --skip-rrna --skip-trna --skip-plot --force  plasmid_query.fa

4. Metadata files:

• bact_meta.tsv - data.table with strain, ST, KL, Year, and Area
• meta - PDG000000012.1904.metadata.tsv (from NCBI Pathogen Watch)
• plasmids.repr.clu - Reference plasmid cluster file (from Klety)
• CARD_AMR_clustered.csv - Carbapenemase gene definitions (from Kleborate)

Input File Formats

BLAST output format (tab-separated):

filename qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore qcovs qcovhsp qlen slen

MOB-typing output:

sample_id rep_type(s) predicted_mobility size

Bakta annotation:

#Sequence Id Type Start Stop Strand Locus Tag Gene Product DbXrefs

bact_meta tsv:

strain ST KL Year Area

Output Files

Generated Files

1. blast_dt. tsv - Filtered and processed BLAST alignments
2. blast_map.tsv - Summary of BLAST mappings with coverage statistics
3. blast_map_f.tsv - Filtered BLAST mappings (complete genomes, coverage >= 80%)
4. evidence.txt - List of strains with fusion evidence
5. blast_dt_list.txt - Detailed BLAST data for plotted comparisons
6. hv_[cluster]_[strain].pdf - Circular genome plots showing:

• Reference plasmid gene annotations
• BLAST alignment coverage
• Gene type color coding (resistance, virulence, plasmid genes, IS elements)
• Temporal information (color-coded by year)

Notes

• The script focuses on PC_499 cluster (pLVPK virulence plasmid) fusion events
• Complete genomes and single-contig plasmids are also prioritized for reference
• The script uses IRanges for accurate coverage calculation accounting for overlaps