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Nextclade is a free web-tool that identifies differences between consensus genome(s) that you upload and the SARS-CoV-2 reference genome MN908947.3. When you upload your sequences to Nextclade, you are able to:
- Assess sequence quality - Nextclade will flag your consensus genome if it does not meet a specific set of requirements.
- See where your samples differ from the reference sequence.
- Identify which clade or lineage your sample belongs to based on differences from the reference genome. Nextstrain has grouped variants of SARS-CoV-2 into clades based on specific signature mutations.
- View sample placement in the context of a Nextstrain phylogenetic tree.
- ***Note: Exercise caution when interpreting the Auspice JSON v2 file generated by Nextclade. Nextclade's algorithms are only meant for quick assessment of sequences. Nextclade is not a replacement for full analysis with the main Nextstrain pipeline. The phylogenetic trees built in Nextclade are not run through the Augur pipeline and therefore are not as rigorous as the phylogenetic trees built in Nextstrain. However, Nextclade can be used as a first pass to understand where your samples fall on the tree.
Check out the Nextstrain blog to read more about SARS-CoV-2 clades!
Uploading to Nextclade
- On the Consensus Genome tab of your project, choose the samples you would like to upload to Nextclade by clicking the checkbox to the left of a sample name.
- Click on the Nextclade button .
- Choose the Nextclade reference tree you would like to see your samples grafted onto. There are two options:
- Nextstrain global tree: Nextclade will provide their global reference tree by default. This is the option you should choose if you do not have your own JSON file.
- If you have built your own tree through the Augur pipeline, you can upload the JSON file here.
- Confirm that you would like to send your data to Nextclade.
- You will be redirected to Nextclade where you can view your results.
There are 4 QC checks that Nextclade performs.
- Missing data- the number of Ns in the consensus genome
- Mixed sites- the number of ambiguous bases (Non A, T, C, or G).
- Private mutations- positions that do not match the reference genome.
- Mutation clusters- clusters of 6 or more mutations in 100 bases (excluding known clusters).
- Frame shifts
- Stop codons
Viewing Nextclade’s phylogenetic tree
*Note: the phylogenetic trees built in Nextclade will give you an idea of where your sequences fall on the tree (either the global Nextstrain tree or the one that you provided). However, the consensus genomes you provide are not processed through the Augur pipeline as is done on Nexstrain. Exercise caution when interpreting the Auspice JSON v2 file generated by Nextclade. Nextclade's algorithms are only meant for quick assessment of sequences. Nextclade is not a replacement for full analysis with the main Nextstrain pipeline.
There are two ways to view your samples grafted onto the reference tree:
- View the tree within Nexclade. This is the simplest way to view the tree, but you will not be able to view associated metadata.
- View the tree in Auspice. This method is great if you have metadata that you would like to view on the tree. Note that all metadata on Auspice is visualized client-side in the browser. See their note about privacy here. Don’t worry- it is still simple!
Option 1: Viewing the tree within Nextclade
Viewing the tree within Nextclade is as simple as clicking ‘Show Tree’ on the results page. The sequences that have been newly added will be highlighted in red.
Option 2: Viewing the tree in Auspice
Auspice is an open-source interactive tool that displays phylogenetic data.
If you would like to view the tree with your metadata, you can do the following:
- In Nextclade, click on the dropdown next to ‘Export to CSV.’
- Choose ‘Export to Auspice’
- An auspice.json file will be generated, which can be dropped onto Auspice here.
- Directions to add metadata to the tree in Auspice can be found here:
- How to Add metadata to your tree in Auspice
- We recommend creating the metadata file in excel and saving it as a CSV or using the metadata CSV file from CZ ID.
If you are interested in Nextstrain, documentation can be found here. There is plenty of excellent documentation, including ‘how to interpret the phylogenetic tree’.
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