Report generation and quality control
Viralgenie's report and result interpreation heavily relies on MultiQC. MultiQC is a tool to create a single report from multiple analysis results. It is designed to be used with a wide range of bioinformatics tools and is compatible with a wide range of data formats. Almost all tools are summarised within the MultiQC report that have interactive plots and data tables.
Tip
Complete output descriptions of files and images can be found in the output section.
Within the multiqc report, viralgenie provides a number of custom tables based consensus genome quality control data. These tools are:
- QUAST: QUAST is a quality assessment tool for genome assemblies. It calculates various metrics such as N50, L50, number of contigs, and total length.
- CheckV: CheckV is a tool for assessing the quality of metagenome-assembled viral genomes. It calculates various metrics such as completeness, contamination, and strain heterogeneity.
- blastn: BLAST is a tool for comparing primary biological sequence information. It calculates the similarity between the consensus genome and the reference genome.
- mmseqs-search: MMseqs is a ultra fast and sensitive search tool for protein and nucleotide databases. Viralgenie uses MMseqs to annotate the consensus genomes and assign them a species name, segment name, expected host etc.
- mafft - not included in MultiQC: MAFFT is a multiple sequence alignment program.
Consensus genome quality control can be skipped with
--skip_consensus_qc.
QUAST
QUAST is a quality assessment tool for genome assemblies. It calculates various metrics such as N50, L50, number of contigs, and total length. However, in the summary table it is mainly used to get the number of ambigous bases in the consensus genome.
Quast can be skipped with
--skip_quast.
CheckV
CheckV is a tool for assessing the quality of metagenome-assembled viral genomes. It calculates various metrics such as completeness, contamination, and strain heterogeneity. CheckV estimates completeness by comparing sequences with a large database of complete viral genomes, metagenomes, metatranscriptomes and metaviromes
Incomplete genomes for segmented viruses
Checkv estimates the completeness of virus based on the all genome segments. If a virus has multiple segments, the completeness of the virus is calculated based on the length of the concatenated segments. For example, Lassa virus has 2 segments L: 7.2kb and S: 3.4kb. The completeness of the virus is calculated based on the length of the concatenated segments (7.2kb + 3.4kb = 10.6kb) and so if the generated consensus genome of the L segment is 7.1kb it will report the completeness as 7.1/10.6 ~ 67%.
Checkv can be skipped with
--skip_checkv.
BLASTn
blastn is a tool for comparing primary biological sequence information. It calculates the similarity between the consensus genome and the reference genome. The similarity is calculated based on the number of identical bases between the two sequences. Viralgenie uses blastn to compare the sequences against the supplied --reference_pool dataset.
Blastn can be skipped with
--skip_blast_qc.
MMseqs-search
MMseqs-search is a ultra fast and sensitive search tool for protein and nucleotide databases. Viralgenie uses MMseqs to search the consensus genomes in a annotated database, like Virousarus (see also defining your own custom annotation database), and uses the annotation data of the best hit to assign the consensus genome a species name, segment name, expected host and any other metadata that is embedded within the database. This allows viralgenie in addition to the blast search of reference pool hits to compare the generated consensus genomes a species & segment level.
Info
MMseqs was used for the annotation step instead of blast because of the ability to query using a tblastx search for highly diverging viruses while supplying a nucleotide annotation database. To specify another type of search (e.g. blastp, blastx, etc.), please refer to the parameters consensus-qc section.
MMseqs-search can be skipped with
--skip_annotation.
MAFFT
MAFFT is a multiple sequence alignment program. It is used to align the following genomic data:
- The final consensus genome
- The identified reference genome from --reference_pool
- The denovo contigs from each assembler (that constituted the final consensus genome)
- Each consensus genome from the iterative refinement steps.
MAFFT can be skipped with
--skip_alignment_qc.
MultiQC
MultiQC is a tool to create a single report with interactive plots for multiple bioinformatics analyses across many samples.
Reports are generated by scanning given directories for recognised log files. These are parsed and a single HTML report is generated summarising the statistics for all logs found. MultiQC reports can describe multiple analysis steps and large numbers of samples within a single plot, and multiple analysis tools making it ideal for routine fast quality control.
Multiqc is ran twice, as the first run is to extract data from all of the tools used so they can be summarised in a multiple tables. These tables can be customised with the argument --custom_table_headers where a yml file, shows which tools need to be included in the summary table in addition to blast, checkv, quast, and mmseqs (annotation).