Genome annotation

A tab separated table describing the genes of the virus (name, frame, position, etc.)

The annotation is required for codon-aware alignment, for translation of CDS (CoDing Sequences), and for calling of amino acid mutations. Without annotation (sometimes called genemap), peptide sequences will not be output and amino acid mutations will not be detected. Without annotation the nucleotide alignment step will not be informed by codon information (see: Algorithm: Sequence alignment and Algorithm: Translation).

Accepted formats: GFF3.

Since version 3, Nextclade supports multi-fragment CDSs which enable the correct translation of complex features including programmed ribosomal slippage (e.g. ORF1ab in SARS-CoV-2), genes crossing the origin of a circular genome (e.g. Hepatitis B virus) and CDS that require splicing (e.g. HIV).

Almost any syntactically correct spec-compliant GFF3 annotation (e.g. downloaded from Genbank) should work. In practice, because GFF3 format allows for great freedom of how to express features as well as how to interpret them, some processing may be required to make it work satisfactory in Nextclade.

At the time of writing, due to historical and technical reasons, Nextclade’s interpretation of GFF3 format has the following deviations from the specification:

• Names of genes are required to be unique

• Names of CDSes are required to be unique

• Names of proteins are required to be unique

The fundamental unit for Nextclade is a single CDS.

When a linked gene and CDS are present (CDSs specify their parents by listing the gene’s ID in the Parent attribute), the gene is effectively ignored for all purposes but display in the web UI. CDS segments are joined if they have the same ID, otherwise they are treated as independent.

Example gene map for SARS-CoV-2:

# seqname	source	feature	start	end	score	strand	frame	attribute
.	.	gene	266	21555	.	+	.	gene=ORF1ab;ID=gene-ORF1ab
.	.	CDS	266	13468	.	+	.	gene=ORF1ab;ID=cds-ORF1ab;Parent=gene-ORF1ab
.	.	CDS	13468	21555	.	+	.	gene=ORF1ab;ID=cds-ORF1ab;Parent=gene-ORF1ab
.	.	CDS	21563	25384	.	+	.	gene=S
.	.	CDS	25393	26220	.	+	.	gene=ORF3a
.	.	CDS	26245	26472	.	+	.	gene=E
.	.	CDS	26523	27191	.	+	.	gene=M
.	.	CDS	27202	27387	.	+	.	gene=ORF6
.	.	CDS	27394	27759	.	+	.	gene=ORF7a
.	.	CDS	27756	27887	.	+	.	gene=ORF7b
.	.	CDS	27894	28259	.	+	.	gene=ORF8
.	.	CDS	28284	28577	.	+	.	gene=ORF9b
.	.	CDS	28274	29533	.	+	.	gene=N

More example annotations can be found in the Nextclade data repository.

Nextclade Web (advanced mode): accepted in “Genome annotation” drag & drop box.

Nextclade CLI flag: --input-annotation/-m

Note: For historical reasons, Nextclade uses gene name when it really means CDS name. The “gene name” is taken from the CDS’s first attribute found in the following list: Gene, gene, gene_name, locus_tag, Name, name, Alias, alias, standard_name, old-name, product, gene_synonym, gb-synonym, acronym, gb-acronym, protein_id, ID.

It is recommended that the gene attribute is used to specify the gene/CDS name.

💡 Nextclade CLI supports file compression and reading from standard input. See section Compression, stdin for more details.