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Interpreting Nextclade quality metrics

article introducing quality metrics for Netxclade

Nextclade is a free and open-source tool for viral genome alignment, mutation calling, clade assignment, quality checks, and phylogenetic positioning. It can rapidly assess viral genomes by running a series of simple, but useful, calculations that can be used to inspect and classify your newly sequenced genomes or as a pre-submission check prior to depositing them in public databases like NCBI and GISAID. It is developed by the same team behind Nextstrain.

Nextclade is available through a command line or web-based interface available at Nextclade website. You can drag and drop a file containing your SC-2 genomes into your browser window, retrieve sequences from a URL, or paste your FASTA sequences into the text field. Nextclade will detect differences between your sequences and a reference sequence (Wuhan-Hu-1/2019), use these differences to classify your sequences into clades/lineages, and flag probable quality issues in your data. All of the analysis takes place entirely within the browser, with no data ever leaving the computer.

The analytical pipeline includes the following steps:

1) Sequence alignment: Using a custom alignment algorithm, sequences are aligned to the reference genome. 2) Translations: Nucleotide sequences are translated into amino acid sequences. 3) Mutation calling: Changes in nucleotides and amino acids are found. 4) Detection of PCR primer changes in binding sites 5) Phylogenetic placement: Sequences are placed on a reference tree, and private mutations are examined. 6) Assignment of Clades: Clades are derived from the tree’s parent node. 7) Quality Control (QC): Metrics for quality control are computed.

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This content is taken from
Wellcome Sanger Institute online course,

A Practical Guide for SARS-CoV-2 Whole Genome Sequencing

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In this example, we examine 61 SC-2 genomes from Peru that were recently sequenced using an Illumina MiSeq instrument. When you press the “run” button, you will immediately see an overview of the results.

Screeshot of Nextclade input page. Red rectangles highlight the “sequence data you’ve added” box and the “run” button. Detailed description in the main text

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Screeshot of Nextclade run output. Detailed description in the main text

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Nextclade uses several quality control indicators to quickly identify problems in your sequences. Bad sequences are red, mediocre ones are yellow, and good ones are white. By hovering your cursor over a sequence’s QC entry, you can get the results of the QC metrics.

Screenshot of Nextclade output. It highlights a box of additional information about the analysed sequences: Missing data, mixed sites, private mutations, mutation clusters, frame shifts and stop codons

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Nextclade also infers the clade/lineage to which a sequence belongs and displays the result in the table. Hovering over the results displays specific information such as the number of mutations, Ns, gaps, insertions, frameshifts, and so on.

Screenshot of Nextclade output. It shows a list of nucleotide substitutions compared to the reference sequence, amino acid substitutions, private mutations and unclassified mutations.

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The alignment can be seen at the right of the window, with missing data shown in grey. You can readily see how missing data segments are scattered around the genome, whether it’s a few large portions clustered in one location or many small missing parts.

You can zoom in on a gene by clicking on it at the bottom, or by selecting it from the dropdown menu at the top. In sequence view, you can see mutations in a specific gene, which can be shown next to its codon translation.

Screenshot of a Nextclade output. It highlights a box that indicates the amino acid and nucleotide changes in a sequence, as well as their correspondent positions in the sequence.