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Toolkit for cleaning and interpreting multiple sequence alignments

Project description



CIAlign is a command line tool which performs various functions to clean and analyse a multiple sequence alignment (MSA).

The tool is designed to be highly customisable, allowing users to specify exactly which functions to run and which settings to use. It is also transparent, generating a clear log file and alignment markup showing exactly how the alignment has changed and what has been removed by which function.

This allows the user to:

  • Remove sources of noise from their MSA

    • Remove insertions which are not present in the majority of sequences
    • Remove sequences below a threshold number of bases or amino acids
    • Crop poorly aligned sequence ends
    • Remove columns containing only gaps
    • Remove sequences above a threshold level percentage of divergence from the majority
  • Generate consensus sequences

  • Visualise alignments

    • Generate image files showing the alignment before and after using CIAlign cleaning functions and showing which columns and rows have been removed
    • Draw sequence logos
    • Visualise coverage at each position in the alignment
  • Analyse alignment statistics

    • Generate a similarity matrix showing the percentage identity between each sequence pair
  • Unalign the alignment


  • python >= 3.6
  • matplotlib >= 2.1.1
  • numpy >= 1.16.3
  • scipy >= 1.3.0


The easiest way to install CIAlign is using pip3:

pip3 install cialign

The current release of CIAlign can also be downloaded directly using this link,

If you download the package directly, you will also need to add the CIAlign directory to your PATH environment variable as described here


Basic Usage

CIAlign --infile INFILE --outfile_stem STEM --inifile my_config.ini


Parameters can be specified in the command line or in a config file using the naming system below.

A template config file is provided in CIAlign/templates/ini_template.txt - edit this file and provide the path to the --inifile argument. If this argument is not provided command line arguments and defaults will be used.

Parameters passed in the command line will take precedence over config file parameters, which take precedence over defaults.

Command help can be accessed by typing CIAlign --help

Parameter Description Default
--infile Path to input alignment file in FASTA format None
--inifile Path to config file None
--outfile_stem Prefix for output files, including the path to the output directory CIAlign
--silent Do not print progress to the screen False
--all Use all available functions with default parameters False
--help Show all available parameters with an explanation None

Beside these main parameters, the use of every function and corresponding thresholds can be specified by the user by adding parameters to the command line or by setting them in the configuration file. Available functions and their parameters will be specified in the following section.

CIAlign always produces a log file, specifying which functions have been run with witch parameters and what has been removed. It also outputs a file that only specifies what has been removed with the original column positions and the sequence names.

Output files:

  • OUTFILE_STEM_log.txt - general log file
  • OUTFILE_STEM_removed.txt - removed columns positions and sequence names text file

Cleaning an MSA

Each of these steps will be performed sequentially in the order specified in the table below.

The "cleaned" alignment after all steps have been performed will be saved as OUTFILE_STEM_cleaned.fasta

Parameter Description Default Value
--remove_divergent Remove sequences with <= N proportion of positions at which the most common base / amino acid in the alignment is present False
--remove_divergent_minperc Minimum proportion of positions which should be identical to the most common base / amino acid in order to be preserved 0.75
--remove_insertions Remove insertions found in <= 50% of sequences from the alignment False
--insertion_min_size Only remove insertions >= this number of residues 3
--insertion_max_size Only remove insertions <= this number of residues 300
--insertion_min_flank Minimum number of bases on either side of an insertion to classify it as an insertion 5
--crop_ends Crop the ends of sequences if they are poorly aligned False
--crop_ends_mingap_perc Minimum proportion of the sequence length (excluding gaps) that is the threshold for change in gap numbers. 0.05
--remove_short Remove sequences <= N bases / amino acids from the alignment False
--remove_min_length Sequences are removed if they are shorter than this minimum length, excluding gaps. 50
--keep_gaponly Keep gap only columns in the alignment True

Generating a Consensus Sequence

This step generates a consensus sequence based on the cleaned alignment. If no cleaning functions are performed, the consensus will be based on the input alignment. For the "majority" based consensus sequences, where the two most frequent characters are equally common a random character is selected.

Output files:

  • OUTFILE_STEM_consensus.fasta - the consensus sequence only
  • OUTFILE_STEM_with_consensus.fasta - the cleaned alignment plus the consensus
Parameter Description Default
--make_consensus Make a consensus sequence based on the cleaned alignment False
--consensus_type Type of consensus sequence to make - can be majority, to use the most common character at each position in the consensus, even if this is a gap, or majority_nongap, to use the most common non-gap character at each position majority
--consensus_keep_gaps If there are gaps in the consensus (if majority_nongap is used as consensus_type), should these be included in the consensus (True) or should this position in the consensus be deleted (False) False
--consensus_name Name to use for the consensus sequence in the output fasta file consensus

Unalign Alignment

This function simply removes the gaps from the input or output alignment and creates and unaligned file of the sequences.

Output files:

  • OUTFILE_STEM_unaligned_input.fasta - unaligned sequences of input alignment
  • OUTFILE_STEM_unaligned_output.fasta - unaligned sequences of output alignment
Parameter Description Default
--unalign_input Generates a copy of the input alignment with no gaps False
--unalign_output Generates a copy of the output alignment with no gaps False

Visualising Alignments

Each of these functions produces some kind of visualisation of your alignment.

Mini Alignments

These functions produce "mini alignments" - images showing a small representation of your whole alignment, so that gaps and poorly aligned regions are clearly visible.

Output files:

  • OUTFILE_STEM_input.png (or svg, tiff, jpg) - the input alignment
  • OUTFILE_STEM_output.png (or svg, tiff, jpg) - the cleaned output alignment
  • OUTFILE_STEM_markup.png (or svg, tiff, jpg) - the input alignment with deleted rows and columns marked
Parameter Description Default
--plot_input Plot a mini alignment - an image representing the input alignment False
--plot_output Plot a mini alignment - an image representing the output alignment False
--plot_markup Draws the input alignment but with the columns and rows which have been removed by each function marked up in corresponding colours False
--plot_dpi DPI for mini alignments 300
--plot_format Image format for mini alignments - can be png, svg, tiff or jpg png
--plot_width Mini alignment width in inches 5
--plot_height Mini alignment height in inches 3

Sequence logos

These functions draw sequence logos representing your output (cleaned) alignment. If no cleaning functions are specified, the logo will be based on your input alignment.


  • OUTFILE_STEM_logo_bar.png (or svg, tiff, jpg) - the alignment represented as a bar chart
  • OUTFILE_STEM_logo_text.png (or svg, tiff, jpg) - the alignment represented as a standard sequence logo using text
Parameter Description Default
--make_sequence_logo Draw a sequence logo False
--sequence_logo_type Type of sequence logo - bar/text/both bar
--sequence_logo_dpi DPI for sequence logo 300
--sequence_logo_font Font (see NB below) for bases / amino acids in a text based sequence logo monospace
--sequence_logo_nt_per_row Number of bases / amino acids to show per row in the sequence logo, where the logo is too large to show on a single line 50
--sequence_logo_filetype Image file type to use for the sequence logo - can be png, svg, tiff or jpg png

NB: to see available fonts on your system, run CIAlign --list_fonts_only and view CIAlign_fonts.png

Coverage Plots

This function plots the number of non-gap residues at each position in the alignment.

Output file:

  • OUTFILE_STEM_input_coverage.png (or svg, tiff, jpg) - image showing the input alignment coverage
  • OUTFILE_STEM_output_coverage.png (or svg, tiff, jpg) - image showing the output alignment coverage
Parameter Description Default
--plot_coverage_input Plot the coverage of the input MSA False
--plot_coverage_output Plot the coverage of the output MSA False
--plot_coverage_dpi DPI for coverage plot 300
--plot_coverage_height Height for coverage plot (inches) 3
--plot_coverage_width Width for coverage plot (inches) 5
--plot_coverage_colour Colour for coverage plot (hex code or name) #007bf5
--plot_coverage_filetype File type for coverage plot (png, svg, tiff, jpg) png

Analysing Alignment Statistics

These functions provide additional analyses you may wish to perform on your alignment.

Similarity Matrices

Generates a matrix showing the proportion of identical bases / amino acids between each pair of sequences in the MSA.

Output file:

  • OUTFILE_STEM_input_similarity.tsv - similarity matrix for the input file
  • OUTFILE_STEM_output_similarity.tsv - similarity matrix for the output file
Parameter Description Default
--make_similarity_matrix_input Make a similarity matrix for the input alignment False
--make_similarity_matrix_output Make a similarity matrix for the output alignment False
--make_simmatrix_keepgaps Include positions with gaps in either or both sequences in the similarity calculation False
--make_simmatrix_dp Number of decimal places to display in the similarity matrix output file 4
--make_simmatrix_minoverlap Minimum overlap between two sequences to have non-zero similarity in the similarity matrix 1

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