Output files

This section describes the files produced by Nextclade.

You can download these files from Nextclade Web using the “Download” dialog.

Download output

Nextclade CLI writes these files into paths specified with a family of --output* flags.

All outputs

Nextclade CLI, Nextalign CLI flags: --output-all

All possible outputs can be produced using --output-all flag. The default base file name is either “nextalign” or “nextclade” depending on which tool you use. It can be changed using --output-basename flag. A list of outputs can be restricted using --output-selection flag.

⚠️ For CLI users: Note that due to technical limitations of the JSON format, it cannot be streamed entry-by entry, i.e. before writing the output to the file, all entries need to be accumulated in memory. If the JSON results output or tree output is requested (through --output-json, --output-tree or --output-all arguments), for large input data, it can cause very high memory consumption, disk swapping, decreased performance and crashes. Consider removing these outputs for large input data, running on a machine with more RAM, or processing data in smaller chunks.

Aligned nucleotide sequences

Nextclade CLI, Nextalign CLI flags: --output-fasta

Aligned sequences are produced as a result of the Sequence alignment step and are being output in FASTA format. If the CLI flag --include-reference is set, the reference sequence is included as the first entry.

⚠️Note that if alignment or analysis of an individual sequence fails, it is omitted from the output alignment file.

Aligned peptides

Nextclade CLI, Nextalign CLI flags: --output-translations

Aligned peptides are produced as a result of the Translation and peptide alignment step and are being output in FASTA format. There are multiple files, one for each gene. If the CLI flag --include-reference is set, the reference sequence peptide is included as the first entry.

This flag accepts a template string which must contain template argument {gene}.

⚠️ Note that if translation, alignment or analysis of an individual gene fails, the corresponding peptide is omitted from the output translation file.

⚠️ Note that if alignment or analysis of an individual sequence fails, all genes are omitted from the output translation files.

Analysis results

The results of mutation calling, clade assignment, quality control and PCR primer changes can be obtained in either TSV, CSV, JSON, or NDJSON format.

⚠️Note that if alignment or analysis of an individual sequence fails, the alignment and translation it is omitted from the output fasta files (see above), but the corresponding entry is still present in most of the other output files. In this case errors column/field contain details about why the processing failed.


If translation, alignment or analysis of an individual gene fails, the corresponding peptide cannot be analyzed, and therefore no details about aminoacid mutations, deletions, insertions, frame shifts etc. will be available. In this case warning and failiedGenes columns/fields contain details about which genes failed and why.


Care should be taken to check for errors, warnings and failedGenes columns or fields, to avoid treating missing or empty entries incorrectly. For example if and errors column is non-empty in TSV output file, it means that the sequence processing failed completely, and treating the empty substitutions column as if no mutations detected is incorrect.


See descriptions of individual outputs and “Outputs for failed sequences” section below for more details.

The next sections describe each analysis output in more details.

Tabular (CSV/TSV) results

Nextclade CLI flags: --output-csv, --output-tsv

TSV and CSV files are equivalent and only differ in the column delimiter (tabs vs semicolons), for better compatibility with spreadsheet software and data-science packages. Tabular format of TSV/CSV files is somewhat human-friendly and convenient for the immediate inspection and for simple automated processing.

Every row in tabular output corresponds to 1 input sequence. The meaning of columns is described below:

Column name Meaning
seqName Name of the sequence (as provided in the input file)
clade Assigned clade
qc.overallScore Overall quality control score
qc.overallStatus Overall quality control status
totalSubstitutions Total number of detected nucleotide substitutions
totalDeletions Total number of detected nucleotide deletions
totalInsertions Total number of detected nucleotide insertions
totalFrameShifts Total number of detected frame shifts
totalAminoacidSubstitutions Total number of detected aminoacid substitutions
totalAminoacidDeletions Total number of detected aminoacid deletions
totalAminoacidInsertions Total number of detected aminoacid insertions
totalMissing Total number of detected missing nucleotides
totalNonACGTNs Total number of detected ambiguous nucleotides
totalPcrPrimerChanges Total number of nucleotide mutations detected in PCR primer regions
substitutions List of detected nucleotide substitutions
deletions List of detected nucleotide deletion ranges
insertions List of detected inserted nucleotide fragments
privateNucMutations.reversionSubstitutions List of detected private mutations that are reversions to reference
privateNucMutations.labeledSubstitutions List of detected private mutations that are to a genotype that has been labeled in virus_properties.json
privateNucMutations.unlabeledSubstitutions List of detected private mutations that are neither reversions nor labeled
privateNucMutations.totalReversionSubstitutions Total number of private mutations that are reversions to reference
privateNucMutations.totalLabeledSubstitutions Total number of private mutations that are to a genotype that has been labeled in virus_properties.json
privateNucMutations.totalUnlabeledSubstitutions Total number of private mutations that are neither reversions nor labeled
privateNucMutations.totalPrivateSubstitutions Total number of private mutations overall
frameShifts List of detected frame shifts
aaSubstitutions List of detected aminoacid substitutions
aaDeletions List of detected aminoacid deletions
aaInsertions List of detected aminoacid insertions
missing List of detected nucleotide insertions
nonACGTNs List of detected ambiguous nucleotides
pcrPrimerChanges List of detected PCR primer changes
alignmentScore Alignment score
alignmentStart Beginning of the sequenced region
alignmentEnd End of the sequenced region
qc.missingData.missingDataThreshold Threshold that was used for "Missing data" QC rule
qc.missingData.score Score for "Missing data" QC rule
qc.missingData.status Status for "Missing data" QC rule
qc.missingData.totalMissing Total number of missing nucleotides used in "Missing data" QC rule
qc.mixedSites.mixedSitesThreshold Threshold used for "Mixed sites" QC rule
qc.mixedSites.score Score for "Mixed sites" QC rule
qc.mixedSites.status Status for "Mixed sites" QC rule
qc.mixedSites.totalMixedSites Total number of ambiguous nucleotides used for "Mixed sites" QC rule
qc.privateMutations.cutoff Cutoff parameter used for "Private mutations" QC rule
qc.privateMutations.excess Excess parameter used for "Private mutations" QC rule
qc.privateMutations.score Score for "Private mutations" QC rule
qc.privateMutations.status Status for "Private mutations" QC rule
qc.privateMutations.total Weighted sum of private mutations used for "Private mutations" QC rule
qc.snpClusters.clusteredSNPs Clustered SNP detected for "SNP clusters" QC rule
qc.snpClusters.score Score for "SNP clusters" QC rule
qc.snpClusters.status Status for "SNP clusters" QC rule
qc.snpClusters.totalSNPs Total number of SNPs for "SNP clusters" QC rule
qc.frameShifts.frameShifts List of detected frame shifts in "Frame shifts" QC rule (excluding ignored)
qc.frameShifts.totalFrameShifts Total number of detected frame shifts in for "Frame shifts" QC rule (excluding ignored)
qc.frameShifts.frameShiftsIgnored List of frame shifts detected, but ignored due to ignore list
qc.frameShifts.totalFrameShiftsIgnored Total number of frame shifts detected, but ignored due to ignore list
qc.frameShifts.score Score for "Frame shifts" QC rule
qc.frameShifts.status Status for "Frame shifts" QC rule
qc.stopCodons.stopCodons List of detected stop codons in "Stop codons" QC rule
qc.stopCodons.totalStopCodons Total number of detected stop codons in "Stop codons" QC rule
qc.stopCodons.score Score for "Stop codons" QC rule
qc.stopCodons.status Status for "Stop codons" QC rule
isReverseComplement Whether query sequences were transformed using reverse complement operation before alignment
errors List of errors during processing
warnings List of warnings during processing
failedGenes List of genes that failed translation

The table can contain additional columns for every clade-like attribute defined in reference tree in meta.extensions.clade_node_attrs and in the node attributes. For example, the default SARS-CoV-2 datasets define Nextclade_pango attribute which signifies a PANGO lineage assigned by Nextclade (see Nextclade as pango lineage classifier: Methods and Validation).

JSON results

Nextclade CLI flag: --output-json, filename nextclade.json.

JSON results file is best for in-depth automated processing of results. It contains everything tabular files contain, plus more, in a more machine-friendly format.

⚠️ For CLI users: Note that due to technical limitations of the JSON format, it cannot be streamed entry-by entry, i.e. before writing the output to the file, all entries need to be accumulated in memory. If the JSON output is requested (through --output-json or --output-all arguments), for large input data, it can cause very high memory consumption, disk swapping, decreased performance and crashes. Consider removing this output for large input data, running on a machine with more RAM, or processing data in smaller chunks.

⚠️ Beware that JSON results reflect internal state of Nextclade, and use 0-indexed nucleotide and codon positions, whereas CSV and TSV files use 1-indexed positions (widely used in bioinformatics). The reason is, that JSON corresponds more closely to the internal representation and 0-indexing is the default in most programming languages. For example, substitution {refNuc: "C", pos: 2146, queryNuc: "T"} in JSON results corresponds to substitution C2147T in csv and tsv files.

Ranges are inclusive for the start and exclusive for the end. Hence, missing: {begin: 704, end: 726} in JSON results corresponds to missing: 705-726 in CSV/TSV results.

NDJSON results

Nextclade CLI flag: --output-ndjson, filename nextclade.ndjson,

NDJSON results are similar to the JSON results - it combines results and errors arrays from it, and similarly suited well for in-depth automated processing of results. It contains everything tabular files contain, plus more, in a more machine-friendly format. Compared to JSON format, NDJSON can be streamed and piped one line at a time, and does not cause increased memory consumption for large input data.

⚠️ Beware that NDJSON results reflect internal state of Nextclade, and use 0-indexed nucleotide and codon positions, whereas CSV and TSV files use 1-indexed positions (widely used in bioinformatics). The reason is, that JSON corresponds more closely to the internal representation and 0-indexing is the default in most programming languages. For example, substitution {refNuc: "C", pos: 2146, queryNuc: "T"} in JSON results corresponds to substitution C2147T in csv and tsv files.

Ranges are inclusive for the start and exclusive for the end. Hence, missing: {begin: 704, end: 726} in NDJSON results corresponds to missing: 705-726 in CSV/TSV results.

Output phylogenetic tree

Nextclade CLI flags: --output-tree, filename: nextclade.auspice.json.

Output phylogenetic tree. This is the input reference tree, with Query Sequences placed onto it.

The tree if Auspice JSON v2 (description, schema) - this is the same format that is used in Nextstrain. And the same as for the input reference tree.

The tree can be visualized online in auspice.us or in a local instance of Nextstrain Auspice.

⚠️ Note that if alignment or analysis of an individual sequence fails, it cannot participate in phylogenetic placement and is omitted from the output tree.

⚠️ For CLI users: Note that due to technical limitations of the JSON format, it cannot be streamed entry-by entry, i.e. before writing the output to the file, all entries need to be accumulated in memory. If the tree output is requested (through --output-tree or --output-all arguments), for large input data, it can cause very high memory consumption, disk swapping, decreased performance and crashes. Consider removing this output for large input data, running on a machine with more RAM, or processing data in smaller chunks.

Stripped insertions

CLI flag: --output-insertions, filename: nextclade.insertions.csv.

Nextclade strips insertions relative to the reference from aligned query sequences, so that inserted fragments no longer appear in the output sequences. This file contains information about the insertions, in CSV format.

⚠️ This flag is deprecated for Nextclade CLI and exist for compatibility with Nextalign CLI. All information in this file is also available in Nextclade CSV, TSV, JSON and NDJSON outputs.

The file contains the following columns (delimited by commas):

  • seqName - Name of the sequence, as in the input FASTA file

  • insertions - A string containing semicolon-separated insertions. Each insertion is in format <begin>:<seq>, where <begin> is the starting position of the insertion in the aligned sequence, <seq> is the nucleotide sequence fragment that was removed, e.g. "22204:GAGCCAGAA".

  • aaInsertions - String containing semicolon-separated insertions translated to aminoacids. Each insertion is in the format <gene>:<pos>:<seq>, e.g. "S:214:EPE".

List of errors and warnings

CLI flag: --output-errors, filename: nextclade.errors.csv.

A table that, for each sequence, contains a list of warnings (column warnings), errors (column errors) as well as a list of genes affected by error (column failedGenes). The genes listed in this table are omitted from translation, analysis and FASTA outputs.

⚠️ This flag is deprecated for Nextclade CLI and exist for compatibility with Nextalign CLI. All information in this file is also available in Nextclade CSV, TSV, JSON and NDJSON outputs.

Outputs for failed sequences

When processing of a sequence fails for various reasons, not all output files will contain the corresponding entry (due to limitations of file formats):

Output file CLI arg Failed entries?
Aligned nucleotide sequences --output-fasta no
Aligned peptides --output-translations no
Auspice tree JSON --output-tree no
Analysis results CSV --output-csv yes
Analysis results TSV --output-tsv yes
Analysis results NDJSON --output-ndjson yes
Analysis results JSON --output-json yes
Insertions CSV --output-insertions yes
Errors CSV --output-errors yes

Compression of output files and writing to standard output (stdout)

If any of the output filenames ends with one of the supported file extensions: gz, bz2, xz, zstd, it will be transparently compressed. Low compression level is used (roughly corresponds to level “2” for most formats).

If output filename is “-” then the output will be written uncompressed to standard output (stdout).

If a custom compression or other form of post-processing is needed, then you could tell Nextclade/Nextalign to write to stdout and then pipe the stdout to another program. For example:

xzcat input.fasta.xz |
nextalign run -r reference.fasta -m genemap.gff -o - |
xz -9 > aligned.slowly.but.heavily.compressed.fasta.xz
xzcat *.fasta.xz |
nextclade run -D dataset/ --output-tsv=- |
process_nextclade_tsv_further > processed.tsv