Tutorial: Analyzing a Virome Dataset with ViromeXplore

The following tutorial uses a 10,000 read subset of a virome from the human gut (SRR829034). The aim of this tutorial is that the user learns to use the workflows contained in the ViromeXplore software. The demo dataset is automatically downloaded with the github repo.

Quality Control and Classification

Run the following command to perform quality control and viral classification:

nextflow ViromeXplore.nf --pipeline qc_classify --reads "demo/demo_SRR829034_{1,2}.fastq"

The interactive Krona plot can be viewed here:

View interactive Krona plot

The ViromeQC results show an enrichment score of 2.26, indicating that the virome is 2.8× more enriched than a comparable metagenome.

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Viral Assembly

Assemble the viral reads:

nextflow ViromeXplore.nf --pipeline viral_assembly --reads "demo/demo_SRR829034_{1,2}.fastq"

View interactive plot

The fatsp report shows that the Q30 of most bases was above 30 so no reads were removed.

This workflow also creates an assembly fasta file with genomic contigs. A quick inspection revealed a total of 27 contigs.

Identify Viral Sequences

Run the viral identification workflow on the assembled MAGs:

nextflow ViromeXplore.nf --pipeline find_viruses --contigs results/megahit_output/final.contigs.fa

Virsorter2 Results: - 3 sequences classified as viral - 2 = dsDNA phages - 1 = ssDNA virus

Refer to the following table for more details:

seqname	dsDNAphage	NCLDV	RNA	ssDNA	lavidaviridae	max_score	max_score_group	length	hallmark	viral
k77_12	0.987	0.260	0.365	1.000	0.267	1.000	ssDNA	2898	1	60.000
k77_25	0.993	0.253	0.185	0.913	0.280	0.993	dsDNAphage	5029	4	62.500
k77_26	1.000	0.473	0.120	0.980	0.947	1.000	dsDNAphage	6399	2	100.000

Check Viral Genome Quality

Results for CheckV:

2 dsDNA viruses: High quality (100% complete)
ssDNA virus: 55% complete

contig_id	contig_length	proviral_length	aai_expected_length	aai_completeness	aai_confidence	aai_error	aai_num_hits	aai_top_hit	aai_id	aai_af	hmm_completeness_lower	hmm_completeness_upper	hmm_num_hits	kmer_freq
k77_12	2899	NA	5203.194186213554	55.71577566105885	high	1.36078157711096	34	DTR_883679	93.88	92.87	45.29686796215677	66.77545783982293	3	1.0
k77_25	5106	NA	5006.547801713951	100.0	high	1.48247660798039	173	DTR_883655	100.0	98.39	85.48428064735897	99.99999999999999	5	1.01
k77_26	6476	NA	6397.397761926372	100.0	high	1.28739096702271	62	DTR_883654	99.74	93.64	95.88582479210984	100.00000000000001	3	1.01

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Extend Incomplete Genomes

nextflow ViromeXplore.nf --pipeline high_quality_genomes \
  --reads "results/fastp_output/demo_SRR829034_{1,2}.fastp.fq.gz" \
  --contigs results/megahit_output/final.contigs.fa \
  --viral_contigs results/checkv_output/viruses.fna

Log output shows no extension was performed (expected for demo data):

[01/23] Reading contigs and getting the contig end sequences...
[05/23] A total of 3 query contigs were imported.
...
no query was extended, exit! this is normal if you only provide few queries.

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Taxonomic Annotation

Since no high-quality genomes were obtained, we run taxonomy annotation directly on the viral contigs:

nextflow ViromeXplore.nf --pipeline taxonomy_annotation \
  --viral_contigs results/checkv_output/viruses.fna

Taxonomy Results: All viral genomese were successfully classified. - Family: Microviridae

seq_name	length	topology	coordinates	n_genes	genetic_code	virus_score	fdr	n_hallmarks	marker_enrichment	taxonomy
k77_26	6476	DTR	NA	9	11	0.9837	NA	2	14.7373	Viruses;Monodnaviria;Sangervirae;Phixviricota;Malgrandaviricetes;Petitvirales;Microviridae
k77_25	5106	DTR	NA	8	11	0.9827	NA	3	11.1680	Viruses;Monodnaviria;Sangervirae;Phixviricota;Malgrandaviricetes;Petitvirales;Microviridae
k77_12	2899	No terminal repeats	NA	5	11	0.9783	NA	1	4.6856	Viruses;Monodnaviria;Sangervirae;Phixviricota;Malgrandaviricetes;Petitvirales;Microviridae

Functional Annotation: - The eggNOG analysis revealed proteins related to:

Structural molecule activity

ATP binding

Viral process

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Directory structure

Each pipeline step creates its own directory, keeping the workflow organized and reproducible, for example:

results/
│
├── samtools_output/
├── megahit_output/
├── bowtie_output/
├── vsearch_output/
├── eggnog_mapper_output/
├── checkv_output/
├── viromeQC_output/
├── fastp_output/
├── geNomad_output/
├── virsorter_out/
├── cdhit_output/
├── mapping_summary_output/
├── kaiju_output/
└── cobra_output/