Load Data

Introduction

Every load option in the Functional Analysis module creates a functional annotation project, the central results object of the module. Depending on the input, the project starts at a different stage of the analysis, which is reflected by the colour and tags of each row in the Main Sequence Table:

• Loading only sequences produces a project without results (white rows).
• Loading BLAST results produces a project with Blast hits (orange rows, BLASTED).
• Loading InterProScan results produces a project with InterPro domains (violet rows, INTERPRO).
• Loading annotations produces an annotated project (blue rows, ANNOTATED).

The full colour and tag scheme is described in Functional Annotation Project. All the options below are found under Functional Analysis → Load (Figure 1).

Load Sequences

Load Example Sequences

Configuration

Available under Functional Analysis → Load → Load Sequences → Load Example Sequences. No input is required; it loads a bundled example sequence set.

Output

A functional annotation project with the example sequences and no analysis results (white rows), useful to try out the module.

Load Fasta File (.fasta)

Configuration

Available under Functional Analysis → Load → Load Sequences → Load Fasta File (.fasta) (Figure 2).

• FASTA file. A text file with one or more DNA or protein sequences in FASTA format. Accepted extensions are .fasta, .fnn, .faa, .fna, .ffn, and .txt.
• Handle Duplicated IDs. Behaviour when a sequence ID in the file is already present in the project: replace the existing entry (default) or skip it.
• Add the Description. When enabled (default), everything after the first white space in the FASTA header (>Id Description) is imported as the sequence description.

A sequence in FASTA format begins with a single-line header starting with a > character, followed by the sequence lines (one character per residue). Avoid unusual characters in the header, such as & or \, and use N for undetermined positions.

>gi|121664|sp|P00435|GSHC BOVIN GLUTATHIONE PEROXIDASE
MCAAQRSAAALAAAAPRTVYAFSARPLAGGEPFNLSSLRGKVLLIENVASLUGTTVRDYTQMND
LQRLGPRGLVVLGFPCNQFGHQENAKNEEILNCLKYVRPGGG

Output

A functional annotation project containing the sequences, with no analysis results yet: all rows are white. From here, any analysis of the module (Blast, InterProScan, GO Mapping, GO Annotation) can be run.

Load Fasta from Reference + GFF/GTF

Configuration

Available under Functional Analysis → Load → Load Sequences → Load Fasta from Reference + GFF/GTF (Figure 3).

• Reference FASTA. The genome FASTA file. Chromosome names must match the sequence IDs in the GFF/GTF file.
• GFF/GTF file. The annotation file describing the features to extract.
• Feature Level. The feature type to extract, as it appears in the third column of the GFF file (e.g., gene, mRNA, exon, CDS).
• Group and Name by. The attribute from the ninth column of the GFF file used as the sequence name. When exon is selected as the feature level, only exons sharing the same value for this attribute are merged into a single sequence.
• Description Template. A free-text template for the description of each extracted sequence. Placeholders of the form $field$ are replaced with the corresponding GFF field or attribute value (e.g., $type$, $gene$, $protein_id$). Standard field names are: $seqid$, $source$, $type$, $start$, $end$, $score$, $strand$, $phase$.
• Skip Duplicates. When enabled (default), sequences whose name is already present in the project are skipped; when disabled, the earlier entry is overwritten.
• Set to Sense. When enabled (default), sequences on the antisense strand are reverse-complemented so all extracted sequences are in sense orientation.

OmicsBox extracts the feature sequences from the genome using the coordinates in the GFF/GTF file. For further information, see the blog post.

Output

A functional annotation project containing the extracted sequences, with no analysis results yet: all rows are white.

Load Blast Results

Configuration

Available under Functional Analysis → Load → Load Blast Results (Figure 4). A single file or a whole directory of BLAST result files can be selected. The following formats are supported:

• Load Blast Results (XML2/JSON). The current NCBI BLAST formats, which can also be loaded from a .zip file.
• Load Blast XML (Legacy). The older NCBI BLAST XML format.

Both formats share the following parameters:

• Filter by Description. All BLAST hits whose description contains the provided text are removed from the result. Multiple filter terms can be separated by semicolons.
• Number of Blast Hits. The maximum number of hit alignments to retain per query sequence (default: 20; range: 0–500).
• HSP Length Cutoff. Minimum length of the first HSP of a BLAST hit. Hits with only short local alignments below this threshold are excluded (default: 33 amino acids or nucleotides; range: 0–5000).
• HSP-Hit Coverage. Minimum percentage of the hit sequence length that must be covered by the best HSP. For example, a value of 80 requires the aligned HSP to span at least 80 % of the hit sequence length (default: 0; range: 0–100).
• Read / Match sequence names in CLC format. When enabled (default), sequence names are parsed in CLC Workbench format to avoid apparent duplicates when BLAST was run on a FASTA file imported by CLC.
• Handling of duplicated ids. Behaviour when a sequence ID from the result file already exists in the project: replace (default) or skip.

The following additional parameters are available for Load Blast XML (Legacy) only:

• Separator. The character used to split the hit description line (default: |).
• Position. Position (1–9) of the description text within the hit definition after splitting by the separator (default: 5).
• Join Hit ID and Hit Description. When enabled (default), the hit ID tag and the hit description tag are concatenated before parsing.

An XML file stores the data inside tags. An example of a BLAST XML2 file:

<?xml version="1.0"?>
<BlastXML2 xmlns="http://www.ncbi.nlm.nih.gov" ...>
    <xi:include href="0e76513c-1bfa-11ea-ad7e-06dd694a34b4_1.xml"/>
    <xi:include href="0e76513c-1bfa-11ea-ad7e-06dd694a34b4_2.xml"/>
    ...
</BlastXML2>

Output

A functional annotation project with the Blast results: sequences with hits are orange (BLASTED). If only the BLAST results are loaded (without the query sequences), the project has no sequence information and the Show Sequence option is disabled; the sequences can be added afterwards (see Add sequences to the existing OmicsBox project).

Load InterProScan Results

Configuration

Available under Functional Analysis → Load → Load InterProScan Results (Figure 5). It loads InterProScan results saved in XML format, into the current project or a new one.

• InterProScan Results. One or more InterProScan XML result files to import.
• Input format. How the results were produced:
  • Protein. InterProScan was run inside OmicsBox (which translates nucleotide sequences to the longest ORF peptides).
  • Nucleotides. InterProScan was run on nucleotide sequences with the InterProScan binaries.

When Nucleotides is selected, the following additional parameters control ORF detection:

• Longest ORF. When enabled (default), only the longest open reading frame per sequence is used for the domain search.
• Strand. Which strand(s) to search for ORFs: Sense, Antisense, or both (default).
• Min ORF Length. Minimum length (in nucleotides) of an ORF to be considered (default: 100).

Output

A functional annotation project with the InterProScan results: sequences turn violet (INTERPRO) if no other analysis has been run. When loaded into a new project, the sequence information is protein and the Show Sequence option is disabled.

Load Annotations

Load Annotations (.annot)

Configuration

Available under Functional Analysis → Load → Load Annotations → Load Annotations (.annot). It imports existing annotations from a .annot file, a tab-separated file with the schema Seq-Name <tab> GO(s) or EC(s) <tab> Sequence description. Several annotations of the same sequence can be given in one row separated by commas, or in subsequent rows.

• Annotation File. The .annot file to import.
• Validate Annotation. When enabled, the imported annotations are validated against the GO true-path rule to ensure consistency (default: disabled).

Seq1    GO:0001234  glycolipid transfer protein-like
Seq1    GO:0001264,GO:0004567
Seq1    EC:2.1.2.10
Seq2    GO:0001234  sorbitol transporter
Seq3    EC:1.2.4.1, EC:3.1

Output

An annotated functional annotation project: annotated sequences are blue (ANNOTATED). When only the annotations are loaded (without the sequences), the project has no sequence information and the Show Sequence option is disabled; the sequences can be added afterwards (see Add sequences to the existing OmicsBox project).

Load Sequence Data / Annotations

Configuration

Available under Functional Analysis → Load → Load Annotations → Load Sequence Data / Annotations. It reads a list of identifiers and connects to NCBI to retrieve both the sequence information and the annotations.

• Protein ID List File. A tab-separated text file with two columns: the identifier (locus or protein) in the first column and the taxonomy identifier in the second.
• Download Sequence Data. When enabled (default), the amino-acid or nucleotide sequences are retrieved from NCBI for each identifier.
• NCBI API Key. An optional NCBI API key that increases the allowed request rate. Registration is free; it is strongly recommended to provide one when querying large identifier lists.
• Export Taxonomic Information. When enabled (default, requires Download Sequence Data), an additional ID-value list mapping each identifier to its taxonomy ID is created; this list can be used for BLAST database creation.
• Add GO Annotation. When enabled (default), GO terms are retrieved via GO Mapping for each protein and added as annotations.

AT1G15520   3702
AT1G18900   3702
AT5G14970   3702

For further information, see the blog post.

Output

A functional annotation project with the retrieved sequences and their annotations: annotated sequences are blue (ANNOTATED).

Load NetAffy Annotations

Configuration

Available under Functional Analysis → Load → Load Annotations → Load NetAffy Annotations. It imports annotation files provided by Affymetrix.

• NetAffy CSV file. An Affymetrix annotation file in CSV format containing the annotated probe IDs. An example can be downloaded here.
• Validate Annotation. When enabled, the imported annotations are validated against the GO true-path rule to ensure consistency (default: disabled).

Output

An annotated functional annotation project: annotated probes are blue (ANNOTATED). As with the other annotation-only options, no sequence information is loaded and the Show Sequence option is disabled.

Load Data from BioMart

Configuration

Available under Functional Analysis → Load → Load Data from BioMart (Figure 6). It retrieves gene or protein sequences and their annotations directly from Ensembl BioMart from a list of identifiers.

• Origin. The Ensembl division to query: Vertebrates, Metazoa, Plants, Protists, or Fungi.
• Dataset (Mart). The BioMart mart to query (populated from the selected origin).
• Database. The dataset within the selected mart (typically a species-specific dataset).
• Type of ID. The type of identifiers provided (filter attribute, e.g., Ensembl Gene ID, UniProt Accession).
• Retrieve Sequence Data. Whether to retrieve sequence data alongside annotations: No (annotations only, default), Protein, or Coding Sequence.
• Which IDs to Import. The source of identifiers to retrieve: User Input (enter identifiers manually, enables the Text and File options), Whole Genome (retrieve all entries in the dataset), or

For further information, see the blog post.

Output

A functional annotation project with the retrieved sequences and their annotations (blue rows). The Sequence Length Distribution chart is also available.

Add sequences to the existing OmicsBox project

Configuration

When a project was created from BLAST or annotation results only (no sequence information), the corresponding sequences can be added afterwards. Click the arrow next to the Start icon and select Load Sequences (.fasta), then choose Add to the existing project and, on the next page, browse for the FASTA file and select the Replace option (Figure 7). Alternatively, use the File Manager context menu: select two or more projects, right-click, and choose Merge.

Output

The existing project is updated with the sequence information, which enables the Show Sequence option and the sequence-based analyses.