Tutorial

Align sequences and build a tree

Align eight DNA sequences with muscle, infer their tree with FastTree, and check it against the tree they were simulated on. All in your browser.

What you will learn

  • Align several sequences with muscle
  • Infer a tree with FastTree and read it in Newick format
  • Interpret branch lengths and support values
  • Compare sequences with TN93 genetic distances

The data

Eight simulated DNA sequences of 594 to 606 bp in four lineages (A, B, C and D, two sequences each). They evolved along a known tree: A with B, C with D. Small insertions and deletions make their lengths differ. Because it is simulated, you can check the tree you get.

Tools in this tutorial

muscle
Aligns several sequences to one another, inserting gaps so that matching positions line up in columns.
FastTree
Infers an approximately maximum-likelihood phylogenetic tree from an alignment and writes it in Newick format with support values.
TN93
Computes pairwise genetic distances between aligned sequences with the Tamura–Nei 1993 model and lists the pairs closer than a threshold.
Download sequences.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 sequences

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

  2. 02

    Align the sequences

    Search muscle and add Align (muscle); connect the Input to it. The sequences have different lengths because of small insertions and deletions, so they must be aligned before they can be compared position by position. Add an Output after muscle to see the alignment.

  3. 03

    Build the tree

    Select muscle, search fasttree and add Build Tree (FastTree). Connect muscle to it, then add an Output after it.

    Workflow: Input, Align (muscle), an Output for the alignment, Build Tree (FastTree) and an Output for the tree.
  4. 04

    Tell FastTree these are DNA sequences

    Click the FastTree node and switch on nucleotide. Without it, FastTree reads the alignment as protein.

    Build Tree (FastTree) settings with the nucleotide switch turned on.
  5. 05

    Run and look at the alignment

    Press ▶ and click the alignment Output. All eight sequences now have the same length, 606 positions, with - where a sequence has a gap.

    Output panel with the multiple alignment in FASTA format, gaps shown as dashes.
  6. 06

    Read the tree

    Click the tree Output. FastTree writes the tree in Newick format: brackets group sequences, numbers after a colon are branch lengths, and numbers after a closing bracket are support values from 0 to 1.

    Output panel with the tree in Newick format, including branch lengths and support values.

What you should see

(lineage_C_2:0.0064,lineage_C_1:0.0070,(((lineage_B_1:0.0067,lineage_B_2:0.0069)0.978:0.0264,
(lineage_A_1:0.0053,lineage_A_2:0.0082)0.988:0.0253)1.000:0.1325,
(lineage_D_1:0.0066,lineage_D_2:0.0068)0.992:0.0274)0.977:0.0232);

Shortened here to four decimals. Each pair of sequences from the same lineage sits together, A groups with B (support 1.000) and C with D: the tree the sequences were simulated on. The long branch (0.13) separates the two groups. The tree is unrooted, so it starts from an arbitrary point (here lineage C); the groupings are what matter.

The same tree, drawn

Drawn with the root between the two groups. Paste the Newick text into any tree viewer to draw it yourself.

            ┌── lineage_A_1
        ┌───┤
        │   └── lineage_A_2
    ┌───┤
    │   │   ┌── lineage_B_1
    │   └───┤
    │       └── lineage_B_2
────┤
    │       ┌── lineage_C_1
    │   ┌───┤
    │   │   └── lineage_C_2
    └───┤
        │   ┌── lineage_D_1
        └───┤
            └── lineage_D_2

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: which sequences are closest?

Select muscle and add Distance (TN93), connect muscle to it, add an Output and run. TN93 lists only the pairs closer than its default threshold of 0.015 substitutions per site. You should get exactly four pairs, the two sequences of each lineage, at distances of about 0.013 to 0.014. Switch on threshold and raise it to see more distant pairs.

Show the solution
Workflow with Distance (TN93) after muscle; its output lists four pairs, one per lineage, with distances from 0.0133 to 0.0136.
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.