Uploading or Updating data¶
- NGS data (fastq reads) (mandatory)
- Sample metadata (to link each sample to the respective NGS data) (mandatory)
- Reference data (additional user-restricted reference sequences) (optional)
Uploading Sample metadata and NGS data¶
Samples’s metadata and respective NGS data (single-end or paired-end reads in fastq.gz format obtained through widely used technologies, such as Illumina or Ion Torrent) can be uploaded to INSaFLU as a batch (option 1) or individually (option 2):
# Option 1 (Batch)¶
# Option 2 (Individual)¶
Updating Sample metadata¶
You can add/update the Samples’s metadata at any time by simply uploading a table comma-separated (.csv) or tab-separated (.tsv or .txt) table with the updated data (a template file is provided), as follows:
- Go to Samples menu and choose Update metadata, then Load a new file, using one of the provided template files.
The table for Sample metadata update should contain the columns “sample name” (exactly corresponding to the samples to be updated), as well these additional variables (that may not be fulfilled): “data set”, ”vaccine status”, ”week”, ”onset date”, ”collection date”, ”lab reception date”, ”latitude”, ”longitude”. Users are encouraged to include any other columns with metadata variables to be associated with samples (see advantages above).
You cannot update the fastq files. To replace the fastq files associated with a given sample, you need to delete the respective sample and upload all data (metadata plus fastq) again, i.e., create a new sample. You must ensure that the sample to be deleted is not inserted in any Project before deleting it.
Samples names in your account must be unique and only numbers, letters and underscores are allowed.
Uploading Reference data¶
INSaFLU needs reference sequence files to be used for reference-based mapping.
In References menu, INSaFLU provides a set of ready-to-use reference sequences, all publicly available at NCBI (or made available under permission of authors), currently including:
- post-pandemic (2009) vaccine/reference influenza A(H1N1)pdm2009, A(H3N2) and B viruses (from both Northern and Southern hemispheres);
- representative virus of multiple combinations of HA/NA subtypes (i.e., H1N1, H2N2, H5N1, H7N9, etc)
The current list of reference sequences, including loci size and NCBI accession numbers is provided here:
The default reference files (FASTA and GenBank formats) have been prepared to fit amplicon-based schemas capturing the whole CDS of the main eight genes of influenza virus (PB2, PB1, PA, HA, NP, NA, M and NS), such as the wet-lab pre-NGS protocol for influenza whole genome amplification adapted from a RT-PCR assay described by Zhou and colleagues (Zhou et al, 2009, for Influenza A; and Zhou et al, 2014, for Influenza B; Zhou and Wentworth, 2012).
You can download the suggested protocol here:
NO FURTHER ACTIONS ARE NEEDED if you are using the suggested wet-lab pre-NGS protocol and you want to compare your sequences against a reference available at INSaFLU database.
However, you may need to UPLOAD additional reference files to the user-restricted reference database. For instance, you may need to upload the A/H3N2 vaccine reference sequence for the season 2017/2018 (A/Hong Kong/4801/2014 virus), which is not freely available.
- To upload additional references (FASTA format; maximum 50000 bp per file): GO TO References MENU and CHOOSE Add Reference
Important notes - upload new References:
- Multi-FASTA files to be uploaded will typically contain the set of reference sequences that constitute the influenza “whole-genome” sequence of a particular virus (e.g, the combination of the traditional 8 amplicons targeting the 8 eight influenza RNA segments). Still, you are free to upload references files including a specific panel of segments/genes (e.g, segments 4 and 6, which encode the surface proteins HA and NA, respectively)
- Each individual sequence of the multi-FASTA file must have the precise size of each “intra-amplicon” target sequence that you capture by each one of the RT-PCR amplicons.
- INSaFLU automatically annotates the uploaded multi-FASTA sequences upon submission, but, if you prefer, you can also upload (optionally) the respective multi-GenBank file.
## See below a GUIDE to generate additional reference sequences
GUIDE TO GENERATE ADDITIONAL REFERENCE SEQUENCES¶
Please take this guide into account when generating additional reference sequences.
Multi-FASTA files to be upload typically contain the set of reference sequences that constitute the influenza “whole-genome” sequence of a particular virus (e.g, the combination of the traditional 8 amplicons targeting the 8 eight influenza RNA segments). Each individual sequence must have the precise size of each “intra-amplicon” target sequence that you capture by each one of the RT-PCR amplicons.
You may generate your multi-FASTA files in order to fit your amplicon schema by simply adjusting the whole-genome sequences available for download at INSaFLU or at influenza-specific sequence repositories, such as the Influenza Research Database (https://www.fludb.org), NCBI Influenza Virus Resource (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) and EpiFLU/GISAID (https://www.gisaid.org/).
An easy way to handle/generate multi-FASTA files is by opening a text file (e.g., NOTEPAD) and paste individual sequences after each header line. The FASTA IDs (after the ‘>’ character) represent the individual sequence names. For the sake of simplicity, you may designate each sequence as 1, 2, 3, 4, 5, 6 , 7 and 8 (see example), following the traditional influenza segments order (keeping this numerical order is advisable). At the end, you just have to save the multi-FASTA file as “.fasta”. Please avoid symbols or blank spaces in the file names.
INSaFLU automatically annotates uploaded multi-FASTA sequences upon submission, but, if you prefer, you can also upload (optionally) the respective multi-GenBank file. If you upload FASTA and respective GenBank files that have been downloaded from NCBI, please make sure that FASTA ID(s) (after the ‘>’ character) match the name/number that appears in the LOCUS and ACCESSION lines of the GenBank file.
INSaFLU requires reference sequences exclusively composed by non-degenerate bases (i.e. A, T, C, or G). As such, please ensure that all degenerated bases (e.g., R, Y, M, K, S and W) are replaced by non-degenerate sequences before uploading. The choice of the base used in the replacement (e.g., “A” or “G” when replacing an “R”) has no impact on the analysis. It simply means that mutations falling in the replaced nucleotide position will be reported taking into account the reference base selected.
Explore your Sample and Reference databases¶
Samples menu displays all information for all loaded samples (Samples’ names in your account must be unique).
Upon submission, INSaFLU automatically updates samples’ information with reads quality and typing data (automate bioinformatics pipeline modules “Read quality analysis and improvement” and Type and sub-type detection”; see Data analysis in the Documentation).
Just explore the “More info” icon next to each sample.
References menu displays all information for all reference sequences available at your confidential session.
Both FASTA and GenBank files can be downloaded by clicking on the displayed links.
- Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, Wentworth DE. 2009. Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses. J Virol, 83:10309-13.
- Zhou B, Lin X, Wang W, Halpin RA, Bera J, Stockwell TB, Barr IG, Wentworth DE. 2014. Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics. J Clin Microbiol, 52:1330-1337.
- Zhou B, Wentworth DE. 2012. Influenza A virus molecular virology techniques. Methods Mol Biol, 865:175-92.