Working with Multiple References¶
ViroConstrictor can work with multiple references in a single analysis run. This is useful when you are working with samples that contain multiple pathogens, multiple strains of the same pathogen, or when you are unsure which reference to use for a specific sample.
Multiple references should be combined into a single reference FASTA file. This file must contain all the references you wish to use in the analysis. The reference FASTA file can be provided to ViroConstrictor either with the --reference flag or via the samplesheet.
Run a Full Analysis with Multiple References¶
To perform a full analysis with multiple references, simply provide the reference FASTA file using the --reference flag. The file should include all the references you want to use.
By default, the full analysis will be run for each reference in the provided FASTA file, resulting in multiple outputs—one for each reference.
Choose the Best Reference for Each Sample¶
If your sequencing protocol is effective for multiple strains of the same pathogen and you are unsure which reference to use for a particular sample, you can have ViroConstrictor choose the best reference for each sample from a larger set of references.
To enable this, provide all potential references in a single reference FASTA file via the --reference flag or in the samplesheet. Additionally, supply the --match-ref flag or set the Match-ref column in the samplesheet to True.
When enabled, ViroConstrictor will attempt to select the best reference for each sample from those provided.
Example of a Reference FASTA File
Below is an example of a reference FASTA file depicting four subtypes of the measles virus. This file can be provided to ViroConstrictor via the --reference flag or through the samplesheet. With --match-ref enabled or the Match-ref column set to True in the samplesheet, ViroConstrictor will choose the best matching reference for each sample.
>measles_subtype1
atgaaagtaaaactactggtcctg...
>measles_subtype2
atgagtcttctaaccgaggtcgaa...
>measles_subtype3
atgaacccaaatcaaaagataata...
>measles_subtype4
atgagtgacatcgaagccatggcg...
Choose the Best Reference for Each Sample with Segmented Viruses¶
If you have a sequencing protocol that works for multiple strains of the same virus and that virus has a segmented genome (such as influenza), you can have ViroConstrictor choose the best fitting reference for each segment for each sample.
This is achieved by providing a single reference FASTA file that contains all the reference segments (and their variants, if applicable) of the virus. To enable the selection of the best reference for each segment, provide both the --match-ref flag and the --segmented flag, or set both the Match-ref and Segmented columns in the samplesheet to True.
Additionally, extra formatting of the reference FASTA file is required. Each FASTA header should follow the format shown below:
>{personal_identifier} {segment_name}|{segment_subtype}|{extra information}
Example of a Reference FASTA File for Segmented Viruses
Below is an example of a reference FASTA file depicting three segments of three subtypes of the influenza virus.
This file can be provided to ViroConstrictor via the --reference flag or through the samplesheet. With both --match-ref and --segmented enabled (or the corresponding columns set to True in the samplesheet), ViroConstrictor will select the best matching reference for each segment for each sample.
>A.HA_01 HA|H1|H1N1
atgaaagtaaaactactggtcc...
>A.HA_02 HA|H3|H3N2
atgaagactatcattgctttga...
>A.HA_03 HA|H5|H5N1
atgaagactatcattgctttga...>A.MP_01 MP|MP|H1N1
atgagtcttctaaccgaggtcg...
>A.MP_02 MP|MP|H3N2
atgagccttcttaccgaggtcg...
>A.MP_03 MP|MP|H5N1
atgagtcttctaaccgaggtcg...>A.NA_01 NA|N1|H1N1
atgaacccaaatcaaaagataa...
>A.NA_02 NA|N2|H3N2
atgaatccaaatcaaaagataa...
>A.NA_03 NA|N1|H5N1
atgaatccaaatcaaaagataa...
Please note that after choosing the best fitting reference for each segment of the virus for each sample, the FASTA header will be modified to ensure that the results folder is structured correctly. This requires that the {segment_name} format is consistent for every segment variant provided in the reference FASTA file.