We developed ViroMatch to analyze datasets of millions of short next-generation sequence reads to identify viral sequences. The ViroMatch workflow incorporates both nucleotide and translated amino acid sequence alignment against a comprehensive database of viral reference genomes, which allows us the sensitivity to detect highly conserved and divergent viral sequences. Specifically, metagenomic sequences are first screened for putative viral reads by nucleotide alignment with BWA-MEM and translated alignment with Diamond against a database of viral genomes (downloaded from NCBI). This first screen is fast, but the hits include many false positives. Therefore, the putative viral hits are then aligned to the comprehensive NCBI nt nucleotide database using BWA-MEM and the comprehensive NCBI nr protein database using Diamond. Only sequences with an unambiguous alignment to a viral reference are counted as viral hits. Ambiguous hits (those that have alignments with similar scores to viruses and human, bacteria, etc.) are not counted. Ambiguous hits include those that map to repetitive regions that are not suitable for determining virus positivity. This pipeline has been used primarily for analysis of vertebrate viruses, including human viruses found in clinical samples.
Please see https://twylie.github.io/viromatch/ for full documentation and detailed installation instructions.
Gather your input sequences for processing. You may provide either (1) a single input unmapped BAM (uBAM) file or (2) paired FASTQ files. If paired FASTQ files are provided, the R1-file should be first followed by the R2-file, space delimited.
The ViroMatch pipeline, and all of its underlying code dependencies, are provided as a Docker image. Once you've installed Docker Desktop on your computer and have downloaded the ViroMatch Docker image, you will be able to run the pipeline containers.
Once you've installed Docker Desktop (and the Docker service is running on your system) you will be able to download and run the ViroMatch Docker image. Specifically, you want the viromatch:latest image.
ViroMatch requires several specific databases (--host, --viralfna, --viralfaa, --nt, --nr) for processing. These databases have been pre-compiled and are available for download.
Be aware the databases are quite large in cumulative size (~860 GB).
Databases are being hosting through Globus Connect. Globus Connect is free to install and use for users at non-profit research and education institutions. You will need to login to Globus to access the databases.
Download ViroMatch Databases Using Globus
We can run ViroMatch via a Docker container using the command line. From a terminal, we would type the following to call the Docker container and run ViroMatch. Arguments will need to reflect your file paths and personal settings in order to run properly on your system.
docker \
container run \
-itd \
-v devViroMatchSMK/t/data:/data \
-v /tmp/myTest:/outdir \
-v devViroMatchSMK/t/ncbi/nt:/nt \
-v devViroMatchSMK/t/ncbi/nr:/nr \
-v devViroMatchSMK/t/viral/viralfna:/viralfna \
-v devViroMatchSMK/t/viral/viralfaa:/viralfaa \
-v devViroMatchSMK/t/host:/host \
-v devViroMatchSMK/t/adaptor:/adaptor \
-v devViroMatchSMK/t/taxonomy:/taxonomy \
twylie/viromatch:latest \
viromatch \
--sampleid 'Sample 1' \
--input /data/test.r1.fastq /data/test.r2.fastq \
--outdir /outdir/myTest \
--nt /nt/nt.fofn \
--nr /nr/nr.fofn \
--viralfna /viralfna/viral_genomes.fasta \
--viralfaa /viralfaa/viral_genomes.dmnd \
--host /host/human.fna \
--adaptor /adaptor/adaptor.fqtrim \
--taxid /taxonomy/taxonomy.tsvUpon completion, ViroMatch will provide reports detailing viral taxonomic classification and quantification. All report files are at the top-level of the --outdir directory provided in the execution command.
Results:
REPORT.nuc_ambiguous_counts.txt
REPORT.nuc_counts.txt
REPORT.trans_nuc_ambiguous_counts.txt
REPORT.trans_nuc_counts.txt