Tutorial

Find the genes in a genome

Predict the protein-coding genes of bacteriophage PhiX174 with Prodigal, then compare them with the official annotation. All in your browser.

What you will learn

  • Predict protein-coding genes with Prodigal
  • Read a tool’s error message and act on it
  • Choose between single-genome and meta mode
  • Compare predicted genes with a published annotation

The data

The genome of bacteriophage PhiX174 (NC_001422.1, 5,386 bp). NCBI annotates 11 genes in it, several of them overlapping, which makes it a good test for a gene finder.

Tools in this tutorial

Prodigal
Finds protein-coding genes in bacterial and phage genomes and writes their coordinates and protein sequences.
Download phix174.fasta (5 KB)

Watch it first

The whole tutorial in under 2 minutes, recorded in the live app. The run is sped up.

Steps

  1. 01

    Load the genome

    Open the Workflow Builder at biochef.app. In File manager, upload phix174.fasta and tick it for the Input node. New to the editor? The map reads tutorial shows each move with screenshots.

  2. 02

    Add Prodigal

    Search prodigal and add Predict Genes (Prodigal); connect the Input to it. Prodigal has three outputs: the gene list (out), the proteins (proteins) and the gene sequences (nucleotides). Add an Output node to each.

    Workflow: Input, Predict Genes (Prodigal) and three Output nodes for the gene list, proteins and nucleotides.
  3. 03

    Run it and read the error

    Press ▶. Nothing comes out. Hover the ⓘ on the Prodigal node: Sequence must be 20000 characters (only 5386 read). Consider running with the -p meta option. By default Prodigal learns the genome’s own gene patterns, and that needs at least 20 kb. PhiX174 is 5.4 kb.

    Prodigal log: Sequence must be 20000 characters (only 5386 read), consider running with the -p meta option.
  4. 04

    Switch to meta mode

    Click the Prodigal node, switch on mode and type meta. In this mode Prodigal uses settings pre-trained on many genomes, which works for short sequences.

    Prodigal settings with mode switched on and set to meta.
  5. 05

    Run again and look at the genes

    Press ▶ and click the first Output. Each CDS entry is one predicted gene with its start and end positions, and a note with its start codon (start_type=ATG) and a confidence score.

    Output with seven CDS entries, from 51..221 to 3981..>5384.
  6. 06

    See the proteins

    Click the second Output: the protein sequence of each gene, one letter per amino acid, with * for the stop codon. The first, 56 amino acids long, is the K protein.

    Output with the predicted protein sequences in FASTA format.

What you should see

CDS  51..221        K
CDS  390..848       D
CDS  848..964       J
CDS  1001..2284     F
CDS  2395..2922     G
CDS  2931..3917     H
CDS  3981..>5384    A (partial)

Prodigal finds 7 of the 11 annotated genes; gene names added here from the NCBI annotation. Six match it exactly. Gene A is marked partial (>5384): the genome is circular and A runs past the end back to the start, which a linear sequence cannot show. The four it misses (C, E, A* and B) lie inside or across other genes, a known limit of gene finders.

Use the finished workflow

Import the finished workflow, data and meta mode included, and press ▶. Remember that an exported workflow contains the data loaded in its Input nodes.

Download the finished workflow (JSON)

Exercise: get the genes as a GFF table

The gene list above is in GenBank format. On the Prodigal node, switch on output_format, type gff and run again. You get the same seven genes as GFF, one per line with tab-separated columns, the format that tools such as bedtools read.

Show the solution
Output in GFF format: seven CDS lines for NC_001422.1 from Prodigal_v2.6.3.
Download the solution workflow (JSON)

From a test to a full run

The browser is the place to build and test a workflow on small data like this. For full datasets, the same workflow file is meant to run next to the data with the BioChef agent, which is in development. See Workflows for how the two fit together.

More tutorials: all BioChef tutorials.

This tutorial is licensed under CC BY 4.0. If you use BioChef in your work, please cite Rosa et al., BMC Bioinformatics 2026. BioChef is listed in bio.tools as biotools:biochef.