2025 Phage Group II Introns

About

Code and data corresponing to the paper:

Group II Introns in Phage Genomes (2025)

Liana Merk, Tom Jones, Sean Eddy

Table of Contents

• Genomes

• Figure 1

• Figure 2

• Figure 3

• Figure 4

• Figure 5

• Figure 6

• How to replicate these figures

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Genomes

• Inphared perl script was used in December 2023, Millard Lab, but genomes from this version are available from the Millard lab website.
• IMG/VR database v4.1
• Bakta and Pharokka were both used for annotation of phage genomes with introns.

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Figure 1

Figure 2

Matplotlib python script to create base of subtype tree, later modified in Illustrator.

To assign subtypes, a mixture of secondary structure prediction and sequence similarity was used. The secondary structure prediction was done by viennaRNA and key structural components were verified by eye. Sequence similarity was done by [nhmmer].

cmsearch -o g2_intron_IB.out -A g2_intron_IB.aout --tblout g2_intron_IB.tblout --verbose -E 10 --cpu 64 /n/eddy_lab/users/lmerk/infernal_builds/g2_introns.cm ./IB.fna 

Figure 3

Secondary structure prediction of MN270259 intron by viennaRNA, then modified in RNAcanvas.

Figure 4

4a RVT tree

Follow the protocol listed in 00README.txt.

Figure 5

5a Polymerase map

• Code

  • In an environment with lovis4u installed, run the following command:

    lovis4u -gb seqs -llp left -snl -mmseqs-off -c A4p2

  Note lovis4u will plot coordinates of tRNA features, so a genbank feature with the intron bounds was added to each genbank file, but is saved as a 'tRNA' feature.

• Data

  • Genomes used (genbank files with introns added)
    • Bacillus phage vB_BpuM-BpSp KT895374.gb
    • Staphylococcus phage MarsHill MW248466.gb
    • Bacillus phage vB_BspM_Internexus MW749003.gb
    • Bacillus phage AR9 NC_031039.gb

5b HEG

CapR and EDxHD/Vsr domains were annotated in genbank files for each homing endonuclease. The HEG_svg.py script creates a baseline plot for the HEGs on the same axis.

5c Host Transfer

• Code

  • In an environment with lovis4u installed, run the following command:

    lovis4u -gb seqs -llp left -snl

  This maintains the surrounding gene labels and performs similarity search with mmseqs.

• Data

  • Genomes used (genbank files with introns added)

    • Top panel:

      • Streptococcus phage phi-SgaBSJ31_rum MN270259_lovis.gb
      • Streptococcus equi subsp. equi strain HTP232 CP133956_lovis.gb

    • Bottom panel:

      • Clostridium phage c-st NC_007581.gb
      • Clostridioides difficile strain DSM 102859 CP020378.gb

Figure 6

Follow the protocol listed in 00README.txt.

Supplementary Table 1

To identify the closest uninterrupted gene, the flanking gene was extracted from the genbank file and used as a query in a pHMMER search against Uniprot downloaded in Nov 2024. The top hit was then reported in Supplementary Table 1. This was run with the command:

phmmer -o ${flank}.out --tblout ${flank}.tblout ${flank}.faa /n/eddy_lab/data/uniprot-2024nov/uniprot.fa