Tutorial

Compare two related genomes

Align the genomes of two related phages with minimap2, measure how similar they are, and discover why a circular genome can align in two pieces. All in your browser.

What you will learn

  • Align a whole genome against another with minimap2
  • Read identity and differences from PAF output
  • Understand why circular genomes can align in pieces
  • See how the choice of reference changes the coordinates

The data

Two real genomes from NCBI: bacteriophage PhiX174 (NC_001422.1) and its close relative S13 (NC_001424.1), both 5,386 bp.

Tools in this tutorial

minimap2
Aligns DNA sequences, from short reads to whole genomes, against a reference. It writes SAM for downstream tools or PAF, a plain table of where each sequence matches.
Download phix174.fasta (5 KB)Download s13.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 two genomes

    Open the Workflow Builder at biochef.app. In File manager, upload phix174.fasta and s13.fasta, and tick s13.fasta for the Input node. New to the editor? The map reads tutorial shows each move with screenshots.

  2. 02

    Align one genome to the other

    Add Align (minimap2): S13 into reads, a second Input with phix174.fasta into ref. PhiX174 is the reference: positions are reported on its coordinates.

  3. 03

    Use the genome-to-genome settings

    Click the minimap2 node. Switch on preset and type asm5, the setting for aligning one assembly to another when they differ by well under 5%, as these two do. Switch on output_cigar so each line also counts the differences (NM). Leave sam_output off to get PAF, a table.

    Align (minimap2) settings with preset set to asm5 and output_cigar switched on.
  4. 04

    Run and read the PAF table

    Add an Output and press ▶. Each line is one aligned block: query name, length, start and end; strand; reference name, length, start and end; matching bases; block length; mapping quality; then tags such as NM.

    Output with two PAF lines comparing S13 with PhiX174.

What you should see

NC_001424.1 5386 1469 5382 + NC_001422.1 5386    0 3913 3828 3913 60 NM:i:85
NC_001424.1 5386   49 1469 + NC_001422.1 5386 3966 5386 1400 1420 60 NM:i:20

Two blocks, not one. Both genomes are circles, but each was written down from a different starting point. S13 from 1470 onwards matches the start of PhiX174, and S13’s first part matches PhiX174’s end. Together: 5,228 identical bases out of 5,333 aligned (about 98%), with 105 differences. PAF counts positions from 0.

Use the finished workflow

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

Download the finished workflow (JSON)

Exercise: swap the reference

Swap the connections so that PhiX174 goes into reads and S13 into ref, and run again. You get the same two blocks and the same numbers, with the coordinates on the other side: PhiX174 0–3913 now lands on S13 1469–5382. The choice of reference decides whose positions you read.

Show the solution
PAF output with PhiX174 as query and S13 as reference: the same two blocks with coordinates swapped.
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.