How to effectively use the 'nextclade' command (with examples)

Nextclade is a bioinformatics tool that plays a crucial role in virus research and public health monitoring by facilitating virus genome alignment, clade assignment, and quality control (QC) checks. It is particularly significant in the context of fast-evolving viruses like SARS-CoV-2, where analyzing viral genomes allows researchers to track mutations and the spread of variants efficiently. With a robust command-line interface, Nextclade offers several functionalities to manage and process viral genomes, making it an invaluable asset for researchers and epidemiologists.

Use case 1: Align sequences to user-provided reference, outputting the alignment to a file

Code:

nextclade run path/to/sequences.fa -r path/to/reference.fa -o path/to/alignment.fa

Motivation:

Aligning sequences to a reference genome is crucial to identify genetic variations and mutations. This alignment helps in determining the provenance of a viral strain, identifying mutations, and monitoring the evolution of the virus. By having an output file, researchers can have a record of the alignments for further analysis and comparisons with other genomic data.

Explanation:

• nextclade run: Initiates the command to perform analysis on the input sequences.
• path/to/sequences.fa: Specifies the file path containing the viral genome sequences you wish to align.
• -r path/to/reference.fa: Defines the path to the reference genome file, against which your sequences are aligned.
• -o path/to/alignment.fa: Indicates the output path where the resulting aligned sequences will be stored.

Example Output:

A file named alignment.fa will be generated containing sequences aligned to the given reference, which can then be used for downstream analyses.

Use case 2: Create a TSV report, auto-downloading the latest dataset

Code:

nextclade run path/to/fasta -d dataset_name -t path/to/report.tsv

Motivation:

Generating a TSV report is essential for systematically capturing and analyzing sequence data and clade assignments. By auto-downloading the latest dataset, users ensure that their analyses are based on the most up-to-date reference data, accounting for new mutations and sequences which might have emerged since the last dataset update.

Explanation:

• path/to/fasta: Represents the input file containing the nucleotide sequences to be analyzed.
• -d dataset_name: Automatically downloads the latest version of the specified reference dataset for the analysis.
• -t path/to/report.tsv: Specifies the file path where the resulting TSV report will be saved, allowing users to easily view and interpret results by listing clades and mutations.

Example Output:

A TSV file named report.tsv is produced, summarizing the findings including clade assignments, mutations, and QC scores.

Use case 3: List all available datasets

Code:

nextclade dataset list

Motivation:

Access to the latest reference datasets is fundamental for accurate genomic analysis. By listing all available datasets, researchers can stay informed about which versions and types of datasets are accessible for their specific virus of interest, ensuring they select the most appropriate data for their analyses.

Explanation:

• This command requires no additional arguments and outputs a list of all datasets available for analysis through Nextclade, aiding in dataset selection.

Example Output:

A comprehensive list of datasets, including their names, versions, and release dates, is displayed in the terminal.

Use case 4: Download the latest SARS-CoV-2 dataset

Code:

nextclade dataset get --name sars-cov-2 --output-dir path/to/directory

Motivation:

Staying current with the latest SARS-CoV-2 datasets is vital for researchers tracking this virus due to its rapid mutation rate. Downloading the latest dataset ensures analyses are aligned with the most recent scientific findings and mutations.

Explanation:

• --name sars-cov-2: Specifies the desired dataset, in this case, the SARS-CoV-2 dataset.
• --output-dir path/to/directory: Determines the directory to save the downloaded dataset files, providing easy access for further analysis.

Example Output:

The latest SARS-CoV-2 dataset is downloaded to the specified directory, ready for use in subsequent analyses.

Use case 5: Use a downloaded dataset, producing all outputs

Code:

nextclade run -D path/to/dataset_dir -O path/to/output_dir path/to/sequences.fasta

Motivation:

Utilizing a local dataset allows for comprehensive analysis under controlled conditions, avoiding dependency on real-time internet access. This setup is particularly beneficial for analyses in isolated environments or when working with proprietary data needing offline processing.

Explanation:

• -D path/to/dataset_dir: Specifies the path to locally stored dataset files, which will be used for sequence analysis.
• -O path/to/output_dir: Output directory where all files generated by the analysis will be saved.
• path/to/sequences.fasta: Indicates the input sequences to be processed using the specified dataset.

Example Output:

All resulting files, including alignment results, reports, and logs, are generated in the designated output directory.

Use case 6: Run on multiple files

Code:

nextclade run -d dataset_name -t path/to/output_tsv -- path/to/input_fasta_1 path/to/input_fasta_2 ...

Motivation:

Analyzing multiple files in a single batch increases efficiency and reduces computational overhead, especially when dealing with large datasets or numerous samples collected from different regions or time points. This capability streamlines workflow by producing a comprehensive output in one go.

Explanation:

• -d dataset_name: Selects the dataset used for analysis.
• -t path/to/output_tsv: Designates the path to save the cumulative output report from all input files.
• --: Signifies the end of options and the start of the input file list.
• path/to/input_fasta_1 path/to/input_fasta_2 ...: Lists all input FASTA files to be included in the batch processing.

Example Output:

A single TSV file is generated, containing aligned results from all the input sequences.

Use case 7: Try reverse complement if sequence does not align

Code:

nextclade run --retry-reverse-complement -d dataset_name -t path/to/output_tsv path/to/input_fasta

Motivation:

Sequences may sometimes be input in the reverse orientation. Automatically retrying the alignment with the reverse complement can resolve alignment issues that might otherwise require manual sequence checking and correction, enhancing the robustness and accuracy of genomic analyses.

Explanation:

• --retry-reverse-complement: Activates the feature to attempt alignment using the reverse complement of sequences if initial alignment fails.
• -d dataset_name: Choice of dataset for the current analysis.
• -t path/to/output_tsv: Output location for the final TSV report, after retry attempts.
• path/to/input_fasta: Path to the input FASTA file comprising sequences for alignment.

Example Output:

A TSV file reflecting corrected alignments, including sequences initially misaligned in their reverse orientation.

Conclusion:

Nextclade emerges as an essential tool in the arsenal of any genomic researcher dealing with viruses, offering a comprehensive suite of features for alignment, clade assignment, and quality control. With the ability to efficiently process sequences leveraging both online and offline datasets, it empowers scientists to maintain an edge in viral genomics research, rapidly adapting to new insights in this fast-paced field. By following the use cases outlined above, users can maximize the tool’s utility, ensuring precise, up-to-date genomic analyses.