Overview

The steps that sphae takes are shown here:

This snakemake workflow was built using Snaketool [https://doi.org/10.1371/journal.pcbi.1010705], to assemble and annotate phage sequences. Currently, this tool is being developed for phage genomes. The steps include,

• Quality control that removes adaptor sequences, low-quality reads and host contamination (optional).
• Assembly
• Contig quality checks; read coverage, viral or not, completeness, and assembly graph components.
• Phage genome annotation

A complete list of programs used for each step is mentioned in the sphae.CITATION file.

If you are new to bioinformatics, here is a tutorial to follow: https://github.com/AnitaTarasenko/sphae/wiki/Sphae-tutorial

Pip install

#creating a new envrionment
conda create -y -n sphae python=3.12
conda activate sphae
#install sphae 
pip install sphae

Conda install

#creating a new environment
conda create -y -n sphae 
conda activate sphae
#install sphae
mamba install sphae

Source Install

Setting up a new conda environment

conda create -n sphae python=3.12
conda activate sphae
conda install -n base -c conda-forge mamba #if you don't already have mamba installed

Steps for installing sphae workflow

#clone sphae repository
git clone https://github.com/linsalrob/sphae.git

#move to sphae folder
cd sphae

#install sphae
pip install -e .

#confirm the workflow is installed by running the below command 
sphae --help

Container Install

You can use the pre-built sphae container with Docker/Singularity/apptainer available here. It is very large as it comes with all the required software pre-installed, so may take a while to download and install.

As an example of installing the sphae .sif file and running sphae v1.4.4 with Singularity:

TMPDIR=<where your tmpdir lives>
IMAGEDIR-<where you want the image to live>

singularity pull --tmpdir=$TMPDIR  --dir $IMAGEDIR docker://quay.io/gbouras13/sphae:1.4.4
singularity exec  sphae_1.4.4.sif sphae --help 
singularity exec  sphae_1.4.4.sif sphae run --help
singularity exec  sphae_1.4.4.sif sphae install --help 

You will still need to install the databases with sphae install as outlined below.

Run the below command,

#Installs the database to default directory, `sphae/workflow/databases`
sphae install

#Install database to specific directory
sphae install --db_dir <directory> 

Install the databases to a directory, sphae/workflow/databases

This workflow requires the

• Pfam35.0 database to run viral_verify for contig classification.
• CheckV database to test for phage completeness
• Pharokka databases
• Phynteny models
• Phold databases

This step requires ~17G of storage

The command sphae run will run QC, assembly and annotation

Commands to run

Only one command needs to be submitted to run all the above steps: QC, assembly and assembly stats

#For illumina reads, place the reads both forward and reverse reads to one directory
#Make sure the fastq reads are saved as {sample_name}_R1.fastq and {sample_name}_R2.fastq or with extensions {sample_name}_R1.fastq.gz
sphae run --input tests/data/illumina-subset --output example -k 

#For nanopore reads, place the reads, one file per sample in a directory
sphae run --input tests/data/nanopore-subset --sequencing longread --output example -k

#For newer ONT sequencing data where polishing is not required, run the command
sphae run --input tests/data/nanopore-subset --sequencing longread --output example -k --no_medaka

#To run either of the commands on the cluster, add --executor slurm to the command. There is a little bit of setup to do here.
#Setup a ~/.config/snakemake/slurm/config.yaml file - https://snakemake.github.io/snakemake-plugin-catalog/plugins/executor/slurm.html#advanced-resource-specifications
#I may have set this workflow to run only slurm right now, will make it more generic soon.
sphae run --input tests/data/nanopore-subset --preprocess longread --output example --profile slurm -k --threads 16

Command to run only annotation steps

#the genomes directory has the already assembled complete genomes
sphae annotate --genome <genomes directory> --output example -k 

Output

Output is saved to example/RESULTS directory. In this directory, there will be four files

• Genome annotations in GenBank format (Phynteny output)
• Genome in fasta format (either the reoriented to terminase output from Pharokka, or assembled viral contigs)
• Circular visualization in png format (Pharokka output)
• Genome summary file

Genome summary file includes the following information to help,

• Sample name
• Length of the genome
• Coding density
• If the assembled contig is circular or not (From checkv)
• Completeness (calculated from CheckV)
• Contamination (calculated from CheckV)
• Taxonomy accession ID (Pharokka output, searches the genome against INPHARED database using mash)
• Taxa mash includes the number of matching hashes of the assembled genome to the accession ID/Taxa name. Higher the matching hash- more likely the genome is related to the taxa predicted
• Gene searches:
  • Whether integrase is found (search for integrase gene in annotations)
  • Whether anti-microbial genes were found (Phold and Pharokka search against AMR database)
  • Whether any virulence factors were found (Pharokka search against virulence gene database)
  • Whether any CRISPR spacers were found (Pharokka search against MinCED database)

1. "Failed during assembly":

   • This message indicates that the assembly process was unsuccessful. It suggests that the assembler could not generate contigs, which are contiguous sequences of DNA, typically representing segments of a genome.
   • To confirm this, you can check the logs located at sphae.out/PROCESSING/assembly/flye/<sample name>/assembly_info.txt or sphae.out/PROCESSING/assembly/megahit/<sample name>/log. These logs should provide details about the error or the step at which the assembly failed.
   • One possible reason for this failure could be insufficient genome coverage, meaning that there were not enough sequencing reads to accurately assemble the genome.

2. "Genome includes multiple contigs, fragmented":

   • This message indicates that the assembly generated numerous short fragments (contigs) instead of a single, contiguous sequence representing a nearly complete phage genome.
   • You can verify this by examining the file sphae.out/PROCESSING/assembly/flye/<sample name>-assembly-stats_flye.csv or sphae.out/PROCESSING/assembly/megahit/<sample name>-assembly-stats_megahit.csv.
   • Each row in these tables represents a contig along with its characteristics. If none of the contigs are identified as viral and do not meet a certain completeness threshold (e.g., greater than 70% completeness), it suggests that the assembly consists of fragmented contigs.
   • Fragmented contigs make it challenging to accurately identify genes. To address this issue, you may need to resequence the phages for better coverage or try using different assembly algorithms.

3. "Good genome coverage but still encountering assembly issues":

   • If you have adequate genome coverage but still face assembly problems, you may consider adjusting the subsampling step in sphae. This step involves randomly selecting a subset of reads to reduce the computational burden.
   • To modify the subsampling parameters, navigate to the config/config.yaml file and update the line under subsample section, for example:

     subsample:
         --bases 1000M
     

   • Increase or decrease the number of bases (e.g., 1000M for 1000 megabases) based on your requirements.
   • After making the changes, rerun sphae and ensure that the updated subsampling parameters are reflected in the sphae.out/sphae.config.yaml file.

4. "What does 'No integrases found ...but Phynteny predicted a few unknown function genes to have some similarity with integrase genes but with low confidence. Maybe a false positive or a novel integrase gene' mean?" This message indicates that while no integrase genes were explicitly identified, the analysis detected certain genes that exhibited similarities to integrase genes. However, these genes were associated with low confidence scores, suggesting a possibility of being false positives or potentially representing novel integrase genes.

   Phynteny, the tool used for this prediction, assigns a confidence score to each gene prediction. If this score falls below a certain threshold (typically 90%), the gene remains classified as having an unknown function. To further investigate these genes, advanced techniques such as folding using tools like ColabFold and Foldseek can be employed. Analyzing the structure of these genes may provide additional insights into their functionality and potential role in biological processes.

5. How do I visualize the phages and gene annotations? To visualize the phages and gene annotations, I recommend using Clinker. First, gather all the sample genbank files from sphae.out/RESULTS and place them in a new directory. Then, execute the clinker command to generate clinker plots, which compare the genes in each genome to each other.

   Additionally, for enhanced visualization, consider running dnaapler on the genomes in fasta format obtained from sphae.out/RESULTS. This step generates reoriented phages that start with terminase genes. Pharokka -> Phold -> Phynteny has to be rerun, and the resulting genbank files can be used for visualization. To perform the annotation steps, run the command sphae annotate --input <reoriented genomes from dnaapler in fasta format directory>

   Please note that dnaapler may fail if terminase genes are not found, particularly when working with novel phages. The reason these steps haven't been added to sphae. If you encounter any challenges during this process, please feel free to leave an issue, and I'll provide improved documentation to assist you further with the command on how to install and run the command different commands.

6. Where are the intermediate files being saved? These files are being saved in sphae.out/PROCESSING. If you need more information on the file structure here, or have ideas of better organization then leave an issue and I will make a note to have more documentation.

7. Just run annotation on already assembled genomes?

   sphae annotate --input <input genomes> This command runs only Pharokka, Phold and Phynteny to annotate the assembled genomes. The results are saved to a new directory labeled sphae.out/annotation.

   Note: Currently, Sphae runs Phold in CPU mode, but efforts are underway to support Phold GPU mode for faster processing of this step.

8. How to change the number of base pairs to subsample for a sample? Run the command sphae config This copies the config file within the workflow to the current directory. Open this file and update the line bases: 10000000 to for instance bases: 300000 Then run sphae run with the command sphae run --input tests/data/illumina-subset --output example -k --config <path to the config file with the change>

If you come across any issues or errors, report them under Issues.