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Matrix style alignment handler for multiple sequence alignments (MSA).

Build Status

This alignment handler makes no assumptions about the underlying sequence object. It supports any nucleotide, amino acid and codon sequences that are lists. Any list with payload or state, can be used (e.g. nucleotide quality score, codon annotation). The only requirement is that the list is Enumerable and can be indexed, i.e. inherit Ruby Enumerable and have the [] method.

Features are:

  • Matrix notation for alignment object
  • Functional style alignment access and editing
  • Support for BioRuby Sequences
  • Support for BioRuby trees and node distance calculation
  • bio-alignment interacts well with BioRuby structures, including sequence objects and alignment/tree parsers
  • Support for textual and HTML output of MSA (planned)
  • Support for Clayton's MAF parser is (planned)

When possible, BioRuby functionality is merged in. For example, by supporting Bio::Sequence objects, standard BioRuby alignment functions, sequence readers and writers can be used. By supporting the BioRuby Tree object, standard BioRuby tree parsers and writers can be used. bio-alignment takes alignment handling with phylogenetic tree support to a new level.

bio-alignment is based on Pjotr's experience designing the BioScala Alignment handler and BioRuby's PAML support. Read the Bio::BioAlignment design document for Ruby.


Dependencies are bioruby (bio), bio-bigbio and bio-logger gems. To run the tests you also need to install rspec and cucumber gems.

Bundler is no longer used.

Command line

bio-alignment comes with a command line interface (CLI), which can apply a number of editing functions on an alignment, and generate textual and HTML output. Note that the CLI does not cover the full library. The CLI can be useful for non-Rubyists, pipeline setups, and simply as examples

Remove bridges (columns with mostly gaps) from an alignment

bio-alignment aa-alignment.fa --type aminoacid --edit bridges

Mask islands (short misaligned 'floating' parts in a sequence)

coming soon...

Mask serial mutations

coming soon...

Remove all sequences consisting of mostly gaps (30% informative) and output to FASTA

bio-alignment codon-alignment.fa --type codon --edit info --out fasta

or output codon style

bio-alignment codon-alignment.fa --type codon --edit info --style codon

Remove all sequences containing gaps from an alignment (why would you want to do that?)

bio-alignment codon-alignment.fa --type codon --edit info --perc 100 --out fasta

Section for developers

Codon alignment example

To use the library, load aligned sequences into the Alignment matrix. Here we write an amino acid alignment from a codon aligmment (note codon gaps are represented by '---')

  require 'bio-alignment'
  require 'bigbio' # Fasta reader and writer

  include Bio::BioAlignment
  aln =
fasta ='codon-alignment.fa')
  fasta.each do | rec |
    aln <<, rec.seq)
  # write a matching amino acid alignment
  fasta ='aa-aln.fa')
  aln.rows.each do | row |
    fasta.write(, row.to_aa.to_s)
  # get first codon element of the fourth sequence
  p aln[3][0]

Now add some state - you can define your own row state

  # tell the row to handle state
  # mark the first row for deletion
  aln[0].state =
  if aln.rows[0].state.deleted?
    # do something

Accessing columns

BioAlignment has a module for handling columns in an alignment. As long as the contained sequence objects have the [] and length methods, they can lazily be iterated by column. To get a column and iterate it

  column = aln.columns[3]
  column.each do |element|
    p element

Now add some state - you can define your own column state

  aln.columns[3].state =
  if aln.columns[3].state.deleted?
    # do something

BioRuby Sequence objects

BioAlignment supports adding BioRuby's Bio::Sequence objects:

  require 'bio'  # BioRuby
  require 'bio-alignment'
  require 'bio-alignment/bioruby' # make Bio::Sequence enumerable
  include Bio::BioAlignment

  aln =
  aln <<"atgcatgcaaaa")
  aln <<"atg---tcaaaa")

or use BioRuby's flat file reader

  aln = do |entry|
    aln << entry

and, the other way, we can transform BioAlignment into BioRuby's Bio::Alignment and use BioRuby functions

  bioruby_aln = aln.to_bioruby_alignment

Note that native BioRuby objects may not always work. In the first case, using Bio::Sequence::NA, no ID is passed in, so each sequence is labeled 'id?'. In the second case BioRuby's FlatFile returns a FastaFormat object, this time with ID, but FastaFormat does not support indexing. In general, it is recommended to stay with the bio-alignment Sequence classes (or roll your own, as long as they are Enumerable).


A protein (amino acid) to nucleotide alignment would first load the sequences and align them. Writing a (simple) version of pal2nal would be something like

  fasta3 ='nt-aln.fa')
  aln.each_with_index do | aaseq, i |
    ntseq = aln2.sequences[i] ==
    codonseq =, ntseq.seq)
    codon_pos = 0
    result = []
    aaseq.each do | aa |
      result <<
          codon_pos += 1
    fasta3.write(, result.join(''))

We included a version (of course). With amino acid aa_aln and nucleotide nt_aln loaded, the library version of pal2nal includes validation

  aln = aa_aln.pal2nal(nt_aln, :codon_table => 3, :do_validate => true)

resulting in the codon alignment. A command line pal2nal is also available on installing the gem.


BioAlignment has support for attaching a phylogenetic tree to an alignment, and traversing the tree using an intuitive interface

  newick_tree =  # use BioRuby's tree parser
  tree = aln.attach_tree(newick_tree)         # attach the tree
  # now do stuff with the tree, which has improved bio-alignment support
  root = tree.root
  children = root.children { |n| }.sort.should == ["","seq7"]
  seq7 = children.last
  seq4 = tree.find("seq4")
  seq4.distance(seq7).should == 19.387756600000003
  # find the sequence in the alignment belonging to the node
  print seq4.sequence
  print tree.output_newick                  # BioRuby Newick output

There are methods for finding sibling nodes, splitting the alignment based on the tree, and locating sequences on the same branch. More examples can be found in the tests and features. The underlying implementation of Bio::Tree is that of BioRuby. We have added an OOP layer for traversing the tree by injecting methods into the BioRuby object itself.

Alignment marking/masking/editing

One of the primary reasons for creating BioAlignment is to provide easy ways of editing alignments using a functional style of programming. Primitives are provided which take out much of the plumbing, such as maintaining row/column/element state, and allow copy-on-edit (so no conflicts arise in concurrent code). For example, to walk an alignment by row, and update the row state, you can mark all rows (sequences) which contain many gaps for deletion

  include MarkRows
  mark_rows { |rowstate,row|  # for every row/sequence
    num = row.count { |e| }
    if (num.to_f/row.length) > 0.5
      # this row in the alignment consists mostly of gaps
      rowstate.delete!  # mark row for deletion
    rowstate   # returns the updated row state

next, return a (deep) copy of the original alignment with the rows that are not marked for deletion

  aln2 = aln.rows_where { |row| !row.state.deleted? }

The general idea is that there are many potential ways of selecting rows, and changing some state. The 'mark_rows' function/iterator takes care of the plumbing. All the programmer needs to do is to set the criterion, in this case a gap percentage, and tell the library what state has to change. In this example we only access one row at a time, but you can also access the other rows. You won't be surprised that marking columns looks much the same

  include MarkColumns
  mark_columns { |colstate,col|  # for every column
    num = col.count { |e| }
    if (num.to_f/col.length) > 0.5

''count'' is one of the universal functions that counts elements in a row, column, or alignment.

Next to modifying the state of rows and columns, you can also access the state of alignment elements (i.e. codons, amino acids, nucleotide acids). For example, here we mask every element that has a masked state

  aln = masked_aln.update_each_element { |e| (e.state.masked? ?"X"):e)}

and, here we remove every marked element by turning it into a gap

  aln = marked_aln.update_each_element { |e| (e.state.marked? ?"-"):e)}

''update_each_element'' visits every element in the MSA, and replaces the old with the new.

It is important to note that, instead of directly editing alignments in place, bio-alignment always makes it a two step process. First items are masked/marked through the state of the rows/columns/elements, next the alignment is rewritten using this state. The advantage of using an intermediate state is that the state can be queried for creating (for example) nice output/graphics, using both the original and changed alignments. For example, it is really easy to create a nice output showing which columns were deleted in the original alignment, or which amino acids were masked. Still, methods are available, which hide the two step process, as seen in the next example.

BioAlignment supports many alignment editing features, which are listed here. An edit feature is added at runtime(!) Example:

  require 'bio-alignment/edit/del_bridges'

  aln.extend DelBridges         # mix the module into the object
  aln2 = aln.del_bridges        # execute the alignment editor

where aln2 is a copy of aln with bridging columns deleted.

More examples can be found in the features/edit directory of the source.

See also

For more on the design of bio-alignment, read the Bio::BioAlignment design document.

The API documentation can be found online. For examples see the files in ./spec/*.rb and ./features/*.


If you use this software, please cite one of

This Biogem is published at #bio-alignment


Copyright (c) 2012-2017 Pjotr Prins. See LICENSE.txt for further details.


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