RubberDandelionOmics user tutorial

WORKED EXAMPLE

Case A: from a gene ID to annotation, expression and downloads

Use TkA01G624900 throughout to inspect its reference, annotations, tissue expression and public expression.

1. Open the gene card: confirm TK1151 and negative-strand coordinates; download CDS and protein.

   Open module · Read the steps

2. Inspect ID mapping: the relationship to LXL04_000258 is many-to-one; retain both reference coordinates.

   Open module · Read the steps

3. Read annotation sources, then inspect domain matches and confidence in Pfam.

   Open module · Read the steps

4. Inspect 24 FPKM values from eight tissues and the tissue distribution schematic, then download replicate expression records.

   Open module · Read the steps

5. Select the fixed example matrix, inspect nine TPM expression records at 0, 6 and 24 h and the group means plot, and download TSV.

   Open module · Read the steps

At the end, retain CDS/protein FASTA, tissue-expression TSV and public-expression TSV. Interpret FPKM and TPM separately; do not compare their magnitudes directly.

WORKED EXAMPLE

Case B: from sequence search to a hit gene

Practice how to search a sequence using known-source examples. Run the CDS and protein searches separately; both have been verified on the live website.

1. Obtain the example 1,248-nt CDS or 415-aa protein FASTA.

   Open module · Read the steps

2. Choose the CDS or protein example below and inspect its sequence and fixed parameters.

   Open module · Read the steps

3. Click Run example to submit to the site’s BLAST service; inspect the top source-gene hit.

   Open module · Read the steps

4. Follow the gene-card link in the results to TkA01G624900, then download the current search results TSV.

   Open module · Read the steps

The CDS search returned three hits and the protein search five. Both top hits were the source gene with 100% sequence identity and query coverage. Self-matches demonstrate the retrieval workflow and do not constitute independent functional validation.

CORE ENTRY 1 / 13

Homepage and gene search

Retrieve a gene record in the appropriate reference genome using a known gene ID.

TkA01G624900

Steps

1. Open the homepage and enter the example ID in the gene search box.
2. Submit the query; an exact ID may take you directly to its gene card.
3. Check the returned ID and reference genome version before inspecting gene coordinates and sequences.

Interpreting the results

• TkA identifies TK1151 source IDs; LXL04 identifies OH-1004 source IDs.
• This example opens the TK1151 card for TkA01G624900.

Interpretation limits: Cross-reference mappings are not always one-to-one. Identify the source of your input ID first.

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CORE ENTRY 2 / 13

Gene card and sequences

Inspect coordinates, strand, sequences and related data links.

TkA01G624900

Steps

1. Open the example gene card and verify the resolved ID and reference genome.
2. Read the chromosome, start/end coordinates and strand.
3. Choose CDS, protein or cDNA FASTA according to your task.
4. Follow links to annotation, expression or the genome browser.

Interpreting the results

• The example is at TK1151 Chr01:157738653–157741276 on the negative strand.
• CDS denotes the coding sequence. The example CDS is 1,248 nt long and encodes a 415-aa protein.

Downloads and data retention

Interpretation limits: Gene coordinates depend on the reference genome version. Check the reference source when downloading sequences.

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CORE ENTRY 3 / 13

Gene ID mapping and filtering

Inspect TK1151–OH-1004 mappings and supporting evidence.

TkA01G624900

Steps

1. Open ID mapping and review the references and sources.
2. Filter the table using the example ID.
3. Read the relationship, coverage and synteny fields.
4. Download the current filter; use the full-table link when you need the complete crosswalk.

Interpreting the results

• The example maps to OH-1004 LXL04_000258 with a many-to-one relationship.
• Percentage sequence identity and coverage describe sequence alignments; synteny provides evidence of genomic positional relationships.

Downloads and data retention

Interpretation limits: Complex mappings do not establish biological equivalence between gene records. Retain IDs and coordinates from each reference genome.

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CORE ENTRY 4 / 13

Gene ID conversion

Resolve input gene IDs to gene-level mappings between reference genomes.

TkA01G624900

Steps

1. Enter an ID, or place one ID on each line for a batch.
2. Submit the conversion and compare input and output IDs.
3. Review the mapping category and confidence classification, retaining the reference source of the target ID.
4. Export the current conversion results.

Interpreting the results

• The output includes the relationship between TkA01G624900 and LXL04_000258.
• A gene-level crosswalk does not imply complete transcript or protein equivalence.

Downloads and data retention

Interpretation limits: Review unmapped records and complex mappings individually. Do not infer missing IDs from similar names.

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CORE ENTRY 5 / 13

Public gene expression data

Query sample expression values, group summaries and corresponding plots in a selected public transcriptomic dataset.

TkA01G624900

Steps

1. Select PRJNA745038_TK1151_UNIFORM_V4_TPM and confirm that it is the only selected matrix.
2. Enter the example gene ID and submit the query. Use study categories to filter matrices as needed.
3. Inspect the group expression plot, sample-level values and group statistics, checking treatment, time and units.
4. Download the current-query TSV and retain sample metadata.

Interpreting the results

• The example returns nine sample expression records at 0, 6 and 24 h. Expression values are reported in TPM (transcripts per million).
• Input reference gene IDs and matrix gene-model IDs are recorded separately. The example matrix ID is TKU.015536.
• SD is standard deviation, SE is the standard error of the mean, and n counts expression records in the group. Independent biological replication requires sample metadata.
• The group expression plot displays means as descriptive summaries. Visual differences do not establish statistical significance in differential expression analysis.
• MeJA denotes methyl jasmonate. Archived tissue fields differ from the study description; retain the metadata notice rather than treating laticifer sampling as confirmed.

Downloads and data retention

Interpretation limits: Interpret different studies, reference genomes and TPM/FPKM quantifications separately. Multiple matrices from one study do not constitute additional independent samples.

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CORE ENTRY 6 / 13

Tissue expression values and distribution schematic

Inspect in-house tissue expression values, replicate records and the tissue distribution schematic.

TkA01G624900

Steps

1. Enter the example gene and submit the tissue query.
2. Inspect eight tissue categories, each containing three replicate expression records.
3. Inspect replicate values, tissue summaries and the distribution schematic, checking the legend and color transformation.
4. Download replicate expression values or tissue means and standard deviations (SD), as needed.

Interpreting the results

• The example contains 24 FPKM values (fragments per kilobase of transcript per million mapped fragments). Replicate values and tissue summaries have separate exports.
• The default color transformation is log2(mean FPKM + 1). Read tissue means separately from the transformed color scale.
• Biological versus technical replication remains unspecified for these data. Tissue means and standard deviations are descriptive summaries.

Downloads and data retention

Interpretation limits: In-house tissues and public transcriptomes are separate datasets. Tissue means are not independent replicates.

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CORE ENTRY 7 / 13

Gene annotation

Inspect functional annotations, evidence sources and the interpretation limits of annotation transfer between reference genomes.

TkA01G624900

Steps

1. Enter the example ID and search.
2. Check the input reference, annotation reference and annotation gene ID.
3. Read functional, GO and KEGG-related fields separately.
4. Download the current annotation records.

Interpreting the results

• Source and evidence fields identify how annotations were obtained.
• Transferred annotations retain their original reference source and do not constitute experimental functional validation of the input gene.

Downloads and data retention

Interpretation limits: Descriptions and homology-derived annotations do not replace experimental validation.

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CORE ENTRY 8 / 13

GO / KEGG annotations and statistical enrichment

Inspect homology-transferred annotations of native genes and over-representation analysis (ORA) against an explicit background gene set.

TkA01G624900

Steps

1. Select TK1151 and GO Biological Process (BP). Enter the example gene or one gene ID per line.
2. First inspect input resolution and gene annotations. TK1151 and OH-1004 are analyzed independently without merging genes across references.
3. Inspect eligible foreground/background counts, raw P values and BH-adjusted FDR. A custom background should reflect experimentally detectable genes eligible for selection.
4. Download annotations, the effective background, all statistical results and method sources. Use the real pathway gene-set example for a multiple-gene demonstration.

Interpreting the results

• GO comprises Biological Process (BP), Molecular Function (MF) and Cellular Component (CC). KEGG results retain pathway and KEGG Orthology (KO) identifiers.
• The current implementation uses a one-sided hypergeometric test with separate Benjamini–Hochberg (BH) correction for BP, MF, CC and KEGG.
• k/n is the foreground overlap fraction, and K/N is the background overlap fraction. Fold enrichment is (k/n)/(K/N).
• For this single-gene BP example, the eligible background is N = 11,100 and foreground n = 1. No terms meet FDR ≤ 0.05.
• Plots may include overlapping terms that fail the significance threshold. Interpret FDR, overlap counts and the background definition together.

Downloads and data retention

Interpretation limits: Homology-transferred annotations do not establish orthology or experimental function. Statistical enrichment does not demonstrate pathway activation, regulation or causality.

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CORE ENTRY 9 / 13

Protein domains / Pfam

Inspect Pfam domain matches, protein sequence coordinates and supporting match evidence.

TkA01G624900

Steps

1. Enter the example ID and check the query protein and reference.
2. Read Pfam IDs, names and protein coordinates.
3. Compare domain E-values, coverage fractions and the confidence classifications reported on the page.
4. Download the domain results.

Interpreting the results

• The example contains two domain records: PF00682 (HMGL-like) is classified as high confidence, and PF20500 (DNA-PKcs_N) as low confidence.
• env denotes the predicted domain envelope interval. Blocks and coverage describe its span on the protein sequence, rather than whole-protein sequence identity.
• Confidence levels are computational classifications used by the page, rather than experimental validation levels or probabilities of function.

Downloads and data retention

Interpretation limits: Domain evidence does not establish whole-protein function or experimental validation.

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CORE ENTRY 10 / 13

Nucleotide and protein BLAST

Search the matching reference database with real sequences, then trace hit genes.

TkA01G624900

Steps

1. Download or copy the CDS/protein FASTA supplied in this tutorial.
2. Choose TK1151 and the matching CDS or protein database; select regular mode.
3. Set E-value = 1e-5, maximum hits = 10, and minimum sequence identity and query coverage = 0%.
4. Run the search, inspect hits, open their gene cards and download TSV.

Interpreting the results

• The 1,248-nt CDS and 415-aa protein examples both recover their source gene.
• Percentage sequence identity (Identity) is the fraction of identical residues in an alignment. Query coverage here divides a single high-scoring segment pair (HSP) query-coordinate span by query length.
• The E-value is the expected number of chance alignments scoring at least as highly in the database search. It is not the probability of homology.

Downloads and data retention

Interpretation limits: These are practice searches with known-source sequences. Similarity is not functional or causal validation.

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CORE ENTRY 11 / 13

JBrowse genome browser

Inspect gene structures and neighboring gene annotation tracks within an interval of the selected reference genome.

TK1151 | Chr01:157737000–157743000

Steps

1. Select TK1151 and Chr01, then enter the example interval.
2. Load the interval and wait for browser tracks.
3. Find TkA01G624900 in the gene-model track and inspect strand and exons.
4. Download current view, gene, annotation or structure data as needed.

Interpreting the results

• The example interval spans the negative-strand gene TkA01G624900.
• The application form and embedded browser may use different coordinate conventions. Interpret exported data using their field definitions rather than assuming an indexing origin.

Downloads and data retention

Interpretation limits: Compare coordinates only within one reference, matching chromosome labels to original accessions.

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CORE ENTRY 12 / 13

Data and sequence downloads

Choose files by reference, data type and export scope.

TK1151 / OH-1004 / FASTA / TSV

Steps

1. Open Downloads and check file category, version and original source.
2. Use gene-card exports for one gene and Downloads for complete files.
3. Retain files and sample/source metadata; verify checksums where supplied.
4. Use the dedicated resource page for complete public expression matrices and reproducibility materials.

Interpreting the results

• This tutorial supplies one-gene FASTA and current-query TSV files for small-file practice.
• Whole-reference files, full matrices and current-filter exports have different scopes.

Downloads and data retention

Interpretation limits: Retain sample sources, reference versions and numeric units with downloaded files.

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CORE ENTRY 13 / 13

GWAS candidate gene queries

Use within-family associations for natural rubber content to select candidate genes, then examine annotation and available expression information in gene records for follow-up research.

TkA06G368510

Steps

1. Open the GWAS page to inspect associations, the Manhattan plot and model diagnostics. Select GLM or MLM to change the current model.
2. Open Candidate genes. This view uses a fixed exploratory screening threshold of P≤1×10⁻⁴ and summarizes overlapping or nearest genes for the screened variants.
3. Enter TkA06G368510 in Gene / function / lead variant and submit the query. Optionally select a chromosome to narrow the results.
4. Check gene coordinates, minimum P, in-gene variant count and distance. Select the gene ID to open its record and inspect available annotation, sequences and related data links.
5. Download filtered candidates, candidates from both models or variant–gene evidence as needed. Retain the model, threshold and reference genome information.

Interpreting the results

• Each row represents one gene in the current model. Minimum P and Lead variant identify the smallest P value among the screened variants mapped to that gene and its corresponding variant.
• Variants counts screened variants mapped to the gene at P≤1×10⁻⁴. In-gene variants counts those that overlap the gene interval, rather than all variants in that gene.
• Distance measures the lead variant’s distance from the gene interval; 0 indicates an overlap. Models lists the models meeting the screening threshold and does not represent independent replication.
• In the MLM example, minimum P is approximately 5.15×10⁻⁸, the lead variant is 13,907 bp from the gene interval, no screened variant overlaps the gene, and its annotated function is unknown. It is a nearby candidate, with no rubber-related function established by these observations.
• The two-model candidate download contains 337 model–gene records representing 206 unique genes, including 131 supported by both models. These records do not represent 337 distinct genes.
• The association analysis includes 126 F₁ offspring. Resequencing variant browsing covers 129 samples, comprising 127 offspring and two parents; these are different sample sets.

Downloads and data retention

Interpretation limits: The results are this site’s reanalysis of public CRA009020 resequencing and natural rubber content phenotypes from the original study, and support exploratory association within a single family. P≤1×10⁻⁴ is distinct from FDR or Bonferroni criteria; proximity and support from two models do not establish causality. See Samples & quality and the GWAS methods notes for sample correspondence, model diagnostics and original-study details. Expression or protein records may not be available for every candidate.

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Terminology

• Reference genome: the specified genome assembly used to define gene coordinates, sequences and annotations.
• Annotation transfer: annotations propagated through sequence homology or mapping, with donor and source information retained. Transfer does not constitute experimental validation.
• Foreground gene set: the target genes tested. Background gene set: genes eligible for foreground selection and meeting annotation requirements in the analysis.
• FDR: false discovery rate. This page reports BH-adjusted values, which should be distinguished from raw P values.
• Replication: distinguish biological replicates, technical replicates and pooled libraries using the experimental design. Record counts alone do not establish independent replication.
• FASTA: a sequence text format with identifier headers. TSV: a tab-separated text table format.
• nt denotes nucleotide length, aa amino acid residue length, and h hours.

Common questions

Data sources · Public matrices and reproducibility resources