The terminology in bioinformatics is often ambiguous, with some terms not being defined well and some terms having different meaning, depending on context and research area.

In order to improve understanding of this documentation and of the source code of the project, in this section we try to summarize the terminology used by Nextclade, including possible synonyms. This terminology is not perfect or complete, and some of the definitions are purposefully simplified, to narrow down the scope to the topics relevant for the project.

For clarity, when possible, please use this vocabulary when communicating with Nextclade team.

We will be grateful for contributions to this section.

Reference sequence

Synonyms: Root sequence

The sequence against which the Alignment and Analysis are modelled.

Reference sequence is expected to be mostly complete (no or few unsequenced or missing regions) and unambiguous (no or few no ambiguous nucleotides) and is expected to correspond to the root node of the phylogenetic tree.

The quality of reference sequence is important for the quality of the analysis.

Root sequence

Same as Reference sequence.

The name originates from the Root node of the Reference tree (concept).

Query sequence

Synonyms: Query nucleotide sequence

One of the input nucleotide sequences provided by the user. These are the sequences to be analyzed.

Reference nucleotide

A nucleotide (character) in the Reference sequence.

Query nucleotide

Synonyms: Derived nucleotide

A nucleotide (character) in the Query sequence.


A nucleotide sequence fragment that is read and transcribed by the polymerase. Often, genes contain encoding a CDS. Gene and CDS are often used synonymously.


Abbreviation of Coding Sequence. Nucleotide sequence that is translated by the ribosome and codes for a Peptide.

Codon (concept)

Synonyms: Triplet

A set of 3 consecutive nucleotides, encoding 1 aminoacid.

Codon (position)

Numeric index of the [Codon (concept)] in a Gene.


Synonyms: Aminoacid sequence

Translated nucleotide sequence of a Gene. A sequence consisting of aminoacids.

Query peptide

A Peptide corresponding to one of the Genes in the Query sequence

Reference peptide

A Peptide corresponding to one of the Genes in the Reference sequence

Query aminoacid

Synonyms: Derived aminoacid

Aminoacid in the Query peptide

Reference aminoacid

Aminoacid in the Query peptide

Reference tree (concept)

Phylogenetic tree - the tree diagram showing evolutionary relationships. Every node corresponds to a particular sequence. This tree is to be used as a source of clade annotations and as a target for phylogenetic placement.

Reference tree (file)

The file that encodes the Reference (phylogenetic) tree (concept). Most often refers to the tree files in Auspice JSON v2 format.

Reference node

(not the same as Root node

The node of the original reference tree.

Before Plylogenetic placement all nodes of the tree are the reference nodes (there are no before New nodes yet).

Root node

Root node of the reference tree. This is the parent node for all other nodes.

The root node corresponds to the Reference sequence.

New node

Node on the reference tree that corresponds to a particular Query sequence placed onto the tree during the Plylogenetic placement.

Gene map

(used interchangeably with Genome annotation)

A set of entries describing CDS for a particular virus. This includes names, nucleotide ranges of each CDS. The annotation can also contain other features such as genes or RNA secondary structures.

Alignment (process)

(used interchangeably with Sequence alignment, Nucleotide alignment, Peptide alignment and Aminoacid alignment, depending on the surrounding context)

The process of arranging Query sequence against Reference sequence (or Query peptide against Reference peptide) to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences.

During alignment, the fragments of the query sequence are compared to the fragments of the reference sequence, the similarities are identified and the fragments are repositioned such that to increase similarity. The resulting aligned sequences allow comparisons on nucleotide (or aminoacid) level and to perform further analysis for example deducing mutations and other features of practical interest).

(this definition is adapted with modifications from: wikipedia: Sequence alignment)

See Algorithm: phylogenetic placement for more details.

Alignment (result)

The Query sequence (or Query peptide) after the Alignment (process).

Alignment range

Numeric range of nucleotide positions signifying begin and end of the aligned sequence.


A virus variant, typically one of a several co-circulating. in Nextstrain, clades are defined by their combination of signature mutations.

See also: Wikipedia: Clade

Phylogenetic placement

The process of adding New nodes to the the Reference tree.

See Algorithm: phylogenetic placement for more details.


The process of performing various steps within the Nextclade algorithm.

Branch length

Branches in a phylogenetic tree connect parent and child nodes. Their length corresponds to a distance between parent and child. In general, this distance can have many different qualities and could for example correspond to calendar time, morphological distance, or genetic distance. The latter – genetic distance – is the most common distance metric in phylogenetic trees and is measured as the number of substitutions between the parent and the child sequence. This measure is often normalized by the length of the sequence, converting the number of substitutions into the fraction of positions of the genome that changed along the branch. When probabilistic models of sequence evolution are used, branch length is typically the expected number or fraction of substitutions. Insertions and deletions are often ignored in the calculation of branch length since they can involve a large number of positions and differences between sequences due to insertions and deletions cannot be treated on a position-by-position basis as single nucleotide changes.


Sequence divergence describes the cumulative distance of a node in the tree from the root of the tree and is calculated by summing all length of branches on the path from the root to the node. Annotation of a tree with branch length and divergences is thus equivalent.

Private Mutations

Nextclade introduces the concept of private mutations. These are the differences between a query sequence and the sequence of the most similar node of the reference tree. When attaching the query sequence as a new node in the tree, these private mutations are the mutations that map to the branch from the nearest reference node to the new node corresponding to the query sequence and determine the length of this branch.


Synonyms: Reading frame

A particular way to split nucleotide sequence into triplets. Each frame corresponds to one of the possible translations of a sequence.

See Wikipedia - Reading frame

Frame shift

Shift of the reading frame. In context of Nextclade they can either be:

  • biological corresponding to the real situation in the genome of the pathogen, for example due to insertions and/or deletions of length not divisible by 3, or due to programmed ribosomal frame shift in certain pathogens

  • non-biological, occurring due to defects during sequencing, assembly or processing of query sequences.

See Wikipedia - Ribosomal frameshift