# Multiple Sequence Alignment objects

This chapter describes the older `MultipleSeqAlignment` class and the parsers in `Bio.AlignIO` that parse the output of sequence alignment software, generating `MultipleSeqAlignment` objects. By Multiple Sequence Alignments we mean a collection of multiple sequences which have been aligned together – usually with the insertion of gap characters, and addition of leading or trailing gaps – such that all the sequence strings are the same length. Such an alignment can be regarded as a matrix of letters, where each row is held as a `SeqRecord` object internally.

We will introduce the `MultipleSeqAlignment` object which holds this kind of data, and the `Bio.AlignIO` module for reading and writing them as various file formats (following the design of the `Bio.SeqIO` module from the previous chapter). Note that both `Bio.SeqIO` and `Bio.AlignIO` can read and write sequence alignment files. The appropriate choice will depend largely on what you want to do with the data.

The final part of this chapter is about using common multiple sequence alignment tools like ClustalW and MUSCLE from Python, and parsing the results with Biopython.

## Parsing or Reading Sequence Alignments

We have two functions for reading in sequence alignments, `Bio.AlignIO.read()` and `Bio.AlignIO.parse()` which following the convention introduced in `Bio.SeqIO` are for files containing one or multiple alignments respectively.

Using `Bio.AlignIO.parse()` will return an iterator which gives `MultipleSeqAlignment` objects. Iterators are typically used in a for loop. Examples of situations where you will have multiple different alignments include resampled alignments from the PHYLIP tool `seqboot`, or multiple pairwise alignments from the EMBOSS tools `water` or `needle`, or Bill Pearson’s FASTA tools.

However, in many situations you will be dealing with files which contain only a single alignment. In this case, you should use the `Bio.AlignIO.read()` function which returns a single `MultipleSeqAlignment` object.

Both functions expect two mandatory arguments:

1. The first argument is a handle to read the data from, typically an open file (see Section What the heck is a handle?), or a filename.

2. The second argument is a lower case string specifying the alignment format. As in `Bio.SeqIO` we don’t try and guess the file format for you! See http://biopython.org/wiki/AlignIO for a full listing of supported formats.

There is also an optional `seq_count` argument which is discussed in Section Ambiguous Alignments below for dealing with ambiguous file formats which may contain more than one alignment.

### Single Alignments

As an example, consider the following annotation rich protein alignment in the PFAM or Stockholm file format:

    # STOCKHOLM 1.0
    #=GS COATB_BPIKE/30-81  AC P03620.1
    #=GS COATB_BPIKE/30-81  DR PDB; 1ifl ; 1-52;
    #=GS Q9T0Q8_BPIKE/1-52  AC Q9T0Q8.1
    #=GS COATB_BPI22/32-83  AC P15416.1
    #=GS COATB_BPM13/24-72  AC P69541.1
    #=GS COATB_BPM13/24-72  DR PDB; 2cpb ; 1-49;
    #=GS COATB_BPM13/24-72  DR PDB; 2cps ; 1-49;
    #=GS COATB_BPZJ2/1-49   AC P03618.1
    #=GS Q9T0Q9_BPFD/1-49   AC Q9T0Q9.1
    #=GS Q9T0Q9_BPFD/1-49   DR PDB; 1nh4 A; 1-49;
    #=GS COATB_BPIF1/22-73  AC P03619.2
    #=GS COATB_BPIF1/22-73  DR PDB; 1ifk ; 1-50;
    COATB_BPIKE/30-81             AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA
    #=GR COATB_BPIKE/30-81  SS    -HHHHHHHHHHHHHH--HHHHHHHH--HHHHHHHHHHHHHHHHHHHHH----
    Q9T0Q8_BPIKE/1-52             AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA
    COATB_BPI22/32-83             DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA
    COATB_BPM13/24-72             AEGDDP...AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
    #=GR COATB_BPM13/24-72  SS    ---S-T...CHCHHHHCCCCTCCCTTCHHHHHHHHHHHHHHHHHHHHCTT--
    COATB_BPZJ2/1-49              AEGDDP...AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA
    Q9T0Q9_BPFD/1-49              AEGDDP...AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
    #=GR Q9T0Q9_BPFD/1-49   SS    ------...-HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--
    COATB_BPIF1/22-73             FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA
    #=GR COATB_BPIF1/22-73  SS    XX-HHHH--HHHHHH--HHHHHHH--HHHHHHHHHHHHHHHHHHHHHHH---
    #=GC SS_cons                  XHHHHHHHHHHHHHHHCHHHHHHHHCHHHHHHHHHHHHHHHHHHHHHHHC--
    #=GC seq_cons                 AEssss...AptAhDSLpspAT-hIu.sWshVsslVsAsluIKLFKKFsSKA
    //

This is the seed alignment for the Phage_Coat_Gp8 (PF05371) PFAM entry, downloaded from a now out of date release of PFAM from https://pfam.xfam.org/. We can load this file as follows (assuming it has been saved to disk as “PF05371_seed.sth” in the current working directory):

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")

This code will print out a summary of the alignment:

    >>> print(alignment)
    Alignment with 7 rows and 52 columns
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRL...SKA COATB_BPIKE/30-81
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKL...SRA Q9T0Q8_BPIKE/1-52
    DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRL...SKA COATB_BPI22/32-83
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73

You’ll notice in the above output the sequences have been truncated. We could instead write our own code to format this as we please by iterating over the rows as `SeqRecord` objects:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> print("Alignment length %i" % alignment.get_alignment_length())
    Alignment length 52
    >>> for record in alignment:
    ...     print("%s - %s" % (record.seq, record.id))
    ...
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA - COATB_BPIKE/30-81
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA - Q9T0Q8_BPIKE/1-52
    DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA - COATB_BPI22/32-83
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - COATB_BPM13/24-72
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA - COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - Q9T0Q9_BPFD/1-49
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA - COATB_BPIF1/22-73

You could also call Python’s built-in `format` function on the alignment object to show it in a particular file format – see Section Getting your alignment objects as formatted strings for details.

Did you notice in the raw file above that several of the sequences include database cross-references to the PDB and the associated known secondary structure? Try this:

    >>> for record in alignment:
    ...     if record.dbxrefs:
    ...         print("%s %s" % (record.id, record.dbxrefs))
    ...
    COATB_BPIKE/30-81 ['PDB; 1ifl ; 1-52;']
    COATB_BPM13/24-72 ['PDB; 2cpb ; 1-49;', 'PDB; 2cps ; 1-49;']
    Q9T0Q9_BPFD/1-49 ['PDB; 1nh4 A; 1-49;']
    COATB_BPIF1/22-73 ['PDB; 1ifk ; 1-50;']

To have a look at all the sequence annotation, try this:

    >>> for record in alignment:
    ...     print(record)
    ...

PFAM provide a nice web interface at http://pfam.xfam.org/family/PF05371 which will actually let you download this alignment in several other formats. This is what the file looks like in the FASTA file format:

    >COATB_BPIKE/30-81
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA
    >Q9T0Q8_BPIKE/1-52
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA
    >COATB_BPI22/32-83
    DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA
    >COATB_BPM13/24-72
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
    >COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA
    >Q9T0Q9_BPFD/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
    >COATB_BPIF1/22-73
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA

Note the website should have an option about showing gaps as periods (dots) or dashes, we’ve shown dashes above. Assuming you download and save this as file “PF05371_seed.faa” then you can load it with almost exactly the same code:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.faa", "fasta")
    >>> print(alignment)

All that has changed in this code is the filename and the format string. You’ll get the same output as before, the sequences and record identifiers are the same. However, as you should expect, if you check each `SeqRecord` there is no annotation nor database cross-references because these are not included in the FASTA file format.

Note that rather than using the Sanger website, you could have used `Bio.AlignIO` to convert the original Stockholm format file into a FASTA file yourself (see below).

With any supported file format, you can load an alignment in exactly the same way just by changing the format string. For example, use “phylip” for PHYLIP files, “nexus” for NEXUS files or “emboss” for the alignments output by the EMBOSS tools. There is a full listing on the wiki page (http://biopython.org/wiki/AlignIO) and in the built-in documentation, `Bio.AlignIO`:

    >>> from Bio import AlignIO
    >>> help(AlignIO)

### Multiple Alignments

The previous section focused on reading files containing a single alignment. In general however, files can contain more than one alignment, and to read these files we must use the `Bio.AlignIO.parse()` function.

Suppose you have a small alignment in PHYLIP format:

        5    6
    Alpha     AACAAC
    Beta      AACCCC
    Gamma     ACCAAC
    Delta     CCACCA
    Epsilon   CCAAAC

If you wanted to bootstrap a phylogenetic tree using the PHYLIP tools, one of the steps would be to create a set of many resampled alignments using the tool `bootseq`. This would give output something like this, which has been abbreviated for conciseness:

        5     6
    Alpha     AAACCA
    Beta      AAACCC
    Gamma     ACCCCA
    Delta     CCCAAC
    Epsilon   CCCAAA
        5     6
    Alpha     AAACAA
    Beta      AAACCC
    Gamma     ACCCAA
    Delta     CCCACC
    Epsilon   CCCAAA
        5     6
    Alpha     AAAAAC
    Beta      AAACCC
    Gamma     AACAAC
    Delta     CCCCCA
    Epsilon   CCCAAC
    ...
        5     6
    Alpha     AAAACC
    Beta      ACCCCC
    Gamma     AAAACC
    Delta     CCCCAA
    Epsilon   CAAACC

If you wanted to read this in using `Bio.AlignIO` you could use:

    >>> from Bio import AlignIO
    >>> alignments = AlignIO.parse("resampled.phy", "phylip")
    >>> for alignment in alignments:
    ...     print(alignment)
    ...     print()
    ...

This would give the following output, again abbreviated for display:

    Alignment with 5 rows and 6 columns
    AAACCA Alpha
    AAACCC Beta
    ACCCCA Gamma
    CCCAAC Delta
    CCCAAA Epsilon

    Alignment with 5 rows and 6 columns
    AAACAA Alpha
    AAACCC Beta
    ACCCAA Gamma
    CCCACC Delta
    CCCAAA Epsilon

    Alignment with 5 rows and 6 columns
    AAAAAC Alpha
    AAACCC Beta
    AACAAC Gamma
    CCCCCA Delta
    CCCAAC Epsilon

    ...

    Alignment with 5 rows and 6 columns
    AAAACC Alpha
    ACCCCC Beta
    AAAACC Gamma
    CCCCAA Delta
    CAAACC Epsilon

As with the function `Bio.SeqIO.parse()`, using `Bio.AlignIO.parse()` returns an iterator. If you want to keep all the alignments in memory at once, which will allow you to access them in any order, then turn the iterator into a list:

    >>> from Bio import AlignIO
    >>> alignments = list(AlignIO.parse("resampled.phy", "phylip"))
    >>> last_align = alignments[-1]
    >>> first_align = alignments[0]

### Ambiguous Alignments

Many alignment file formats can explicitly store more than one alignment, and the division between each alignment is clear. However, when a general sequence file format has been used there is no such block structure. The most common such situation is when alignments have been saved in the FASTA file format. For example consider the following:

    >Alpha
    ACTACGACTAGCTCAG--G
    >Beta
    ACTACCGCTAGCTCAGAAG
    >Gamma
    ACTACGGCTAGCACAGAAG
    >Alpha
    ACTACGACTAGCTCAGG--
    >Beta
    ACTACCGCTAGCTCAGAAG
    >Gamma
    ACTACGGCTAGCACAGAAG

This could be a single alignment containing six sequences (with repeated identifiers). Or, judging from the identifiers, this is probably two different alignments each with three sequences, which happen to all have the same length.

What about this next example?

    >Alpha
    ACTACGACTAGCTCAG--G
    >Beta
    ACTACCGCTAGCTCAGAAG
    >Alpha
    ACTACGACTAGCTCAGG--
    >Gamma
    ACTACGGCTAGCACAGAAG
    >Alpha
    ACTACGACTAGCTCAGG--
    >Delta
    ACTACGGCTAGCACAGAAG

Again, this could be a single alignment with six sequences. However this time based on the identifiers we might guess this is three pairwise alignments which by chance have all got the same lengths.

This final example is similar:

    >Alpha
    ACTACGACTAGCTCAG--G
    >XXX
    ACTACCGCTAGCTCAGAAG
    >Alpha
    ACTACGACTAGCTCAGG
    >YYY
    ACTACGGCAAGCACAGG
    >Alpha
    --ACTACGAC--TAGCTCAGG
    >ZZZ
    GGACTACGACAATAGCTCAGG

In this third example, because of the differing lengths, this cannot be treated as a single alignment containing all six records. However, it could be three pairwise alignments.

Clearly trying to store more than one alignment in a FASTA file is not ideal. However, if you are forced to deal with these as input files `Bio.AlignIO` can cope with the most common situation where all the alignments have the same number of records. One example of this is a collection of pairwise alignments, which can be produced by the EMBOSS tools `needle` and `water` – although in this situation, `Bio.AlignIO` should be able to understand their native output using “emboss” as the format string.

To interpret these FASTA examples as several separate alignments, we can use `Bio.AlignIO.parse()` with the optional `seq_count` argument which specifies how many sequences are expected in each alignment (in these examples, 3, 2 and 2 respectively). For example, using the third example as the input data:

    >>> for alignment in AlignIO.parse(handle, "fasta", seq_count=2):
    ...     print("Alignment length %i" % alignment.get_alignment_length())
    ...     for record in alignment:
    ...         print("%s - %s" % (record.seq, record.id))
    ...     print()
    ...

giving:

    Alignment length 19
    ACTACGACTAGCTCAG--G - Alpha
    ACTACCGCTAGCTCAGAAG - XXX

    Alignment length 17
    ACTACGACTAGCTCAGG - Alpha
    ACTACGGCAAGCACAGG - YYY

    Alignment length 21
    --ACTACGAC--TAGCTCAGG - Alpha
    GGACTACGACAATAGCTCAGG - ZZZ

Using `Bio.AlignIO.read()` or `Bio.AlignIO.parse()` without the `seq_count` argument would give a single alignment containing all six records for the first two examples. For the third example, an exception would be raised because the lengths differ preventing them being turned into a single alignment.

If the file format itself has a block structure allowing `Bio.AlignIO` to determine the number of sequences in each alignment directly, then the `seq_count` argument is not needed. If it is supplied, and doesn’t agree with the file contents, an error is raised.

Note that this optional `seq_count` argument assumes each alignment in the file has the same number of sequences. Hypothetically you may come across stranger situations, for example a FASTA file containing several alignments each with a different number of sequences – although I would love to hear of a real world example of this. Assuming you cannot get the data in a nicer file format, there is no straight forward way to deal with this using `Bio.AlignIO`. In this case, you could consider reading in the sequences themselves using `Bio.SeqIO` and batching them together to create the alignments as appropriate.

## Writing Alignments

We’ve talked about using `Bio.AlignIO.read()` and `Bio.AlignIO.parse()` for alignment input (reading files), and now we’ll look at `Bio.AlignIO.write()` which is for alignment output (writing files). This is a function taking three arguments: some `MultipleSeqAlignment` objects (or for backwards compatibility the obsolete `Alignment` objects), a handle or filename to write to, and a sequence format.

Here is an example, where we start by creating a few `MultipleSeqAlignment` objects the hard way (by hand, rather than by loading them from a file). Note we create some `SeqRecord` objects to construct the alignment from.

    >>> from Bio.Seq import Seq
    >>> from Bio.SeqRecord import SeqRecord
    >>> from Bio.Align import MultipleSeqAlignment
    >>> align1 = MultipleSeqAlignment(
    ...     [
    ...         SeqRecord(Seq("ACTGCTAGCTAG"), id="Alpha"),
    ...         SeqRecord(Seq("ACT-CTAGCTAG"), id="Beta"),
    ...         SeqRecord(Seq("ACTGCTAGDTAG"), id="Gamma"),
    ...     ]
    ... )
    >>> align2 = MultipleSeqAlignment(
    ...     [
    ...         SeqRecord(Seq("GTCAGC-AG"), id="Delta"),
    ...         SeqRecord(Seq("GACAGCTAG"), id="Epsilon"),
    ...         SeqRecord(Seq("GTCAGCTAG"), id="Zeta"),
    ...     ]
    ... )
    >>> align3 = MultipleSeqAlignment(
    ...     [
    ...         SeqRecord(Seq("ACTAGTACAGCTG"), id="Eta"),
    ...         SeqRecord(Seq("ACTAGTACAGCT-"), id="Theta"),
    ...         SeqRecord(Seq("-CTACTACAGGTG"), id="Iota"),
    ...     ]
    ... )
    >>> my_alignments = [align1, align2, align3]

Now we have a list of `Alignment` objects, we’ll write them to a PHYLIP format file:

    >>> from Bio import AlignIO
    >>> AlignIO.write(my_alignments, "my_example.phy", "phylip")

And if you open this file in your favorite text editor it should look like this:

     3 12
    Alpha      ACTGCTAGCT AG
    Beta       ACT-CTAGCT AG
    Gamma      ACTGCTAGDT AG
     3 9
    Delta      GTCAGC-AG
    Epislon    GACAGCTAG
    Zeta       GTCAGCTAG
     3 13
    Eta        ACTAGTACAG CTG
    Theta      ACTAGTACAG CT-
    Iota       -CTACTACAG GTG

Its more common to want to load an existing alignment, and save that, perhaps after some simple manipulation like removing certain rows or columns.

Suppose you wanted to know how many alignments the `Bio.AlignIO.write()` function wrote to the handle? If your alignments were in a list like the example above, you could just use `len(my_alignments)`, however you can’t do that when your records come from a generator/iterator. Therefore the `Bio.AlignIO.write()` function returns the number of alignments written to the file.

Note - If you tell the `Bio.AlignIO.write()` function to write to a file that already exists, the old file will be overwritten without any warning.

### Converting between sequence alignment file formats

Converting between sequence alignment file formats with `Bio.AlignIO` works in the same way as converting between sequence file formats with `Bio.SeqIO` (Section Converting between sequence file formats). We load generally the alignment(s) using `Bio.AlignIO.parse()` and then save them using the `Bio.AlignIO.write()` – or just use the `Bio.AlignIO.convert()` helper function.

For this example, we’ll load the PFAM/Stockholm format file used earlier and save it as a Clustal W format file:

    >>> from Bio import AlignIO
    >>> count = AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.aln", "clustal")
    >>> print("Converted %i alignments" % count)
    Converted 1 alignments

Or, using `Bio.AlignIO.parse()` and `Bio.AlignIO.write()`:

    >>> from Bio import AlignIO
    >>> alignments = AlignIO.parse("PF05371_seed.sth", "stockholm")
    >>> count = AlignIO.write(alignments, "PF05371_seed.aln", "clustal")
    >>> print("Converted %i alignments" % count)
    Converted 1 alignments

The `Bio.AlignIO.write()` function expects to be given multiple alignment objects. In the example above we gave it the alignment iterator returned by `Bio.AlignIO.parse()`.

In this case, we know there is only one alignment in the file so we could have used `Bio.AlignIO.read()` instead, but notice we have to pass this alignment to `Bio.AlignIO.write()` as a single element list:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> AlignIO.write([alignment], "PF05371_seed.aln", "clustal")

Either way, you should end up with the same new Clustal W format file “PF05371_seed.aln” with the following content:

    CLUSTAL X (1.81) multiple sequence alignment

    COATB_BPIKE/30-81                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSS
    Q9T0Q8_BPIKE/1-52                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVS
    COATB_BPI22/32-83                   DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSS
    COATB_BPM13/24-72                   AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTS
    COATB_BPZJ2/1-49                    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFAS
    Q9T0Q9_BPFD/1-49                    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTS
    COATB_BPIF1/22-73                   FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVS

    COATB_BPIKE/30-81                   KA
    Q9T0Q8_BPIKE/1-52                   RA
    COATB_BPI22/32-83                   KA
    COATB_BPM13/24-72                   KA
    COATB_BPZJ2/1-49                    KA
    Q9T0Q9_BPFD/1-49                    KA
    COATB_BPIF1/22-73                   RA

Alternatively, you could make a PHYLIP format file which we’ll name “PF05371_seed.phy”:

    >>> from Bio import AlignIO
    >>> AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip")

This time the output looks like this:

     7 52
    COATB_BPIK AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
    Q9T0Q8_BPI AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
    COATB_BPI2 DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
    COATB_BPM1 AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    COATB_BPZJ AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
    Q9T0Q9_BPF AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    COATB_BPIF FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS

               KA
               RA
               KA
               KA
               KA
               KA
               RA

One of the big handicaps of the original PHYLIP alignment file format is that the sequence identifiers are strictly truncated at ten characters. In this example, as you can see the resulting names are still unique - but they are not very readable. As a result, a more relaxed variant of the original PHYLIP format is now quite widely used:

    >>> from Bio import AlignIO
    >>> AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip-relaxed")

This time the output looks like this, using a longer indentation to allow all the identifiers to be given in full:

     7 52
    COATB_BPIKE/30-81  AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
    Q9T0Q8_BPIKE/1-52  AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
    COATB_BPI22/32-83  DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
    COATB_BPM13/24-72  AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    COATB_BPZJ2/1-49   AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
    Q9T0Q9_BPFD/1-49   AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    COATB_BPIF1/22-73  FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS

                       KA
                       RA
                       KA
                       KA
                       KA
                       KA
                       RA

If you have to work with the original strict PHYLIP format, then you may need to compress the identifiers somehow – or assign your own names or numbering system. This following bit of code manipulates the record identifiers before saving the output:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> name_mapping = {}
    >>> for i, record in enumerate(alignment):
    ...     name_mapping[i] = record.id
    ...     record.id = "seq%i" % i
    ...
    >>> print(name_mapping)
    {0: 'COATB_BPIKE/30-81', 1: 'Q9T0Q8_BPIKE/1-52', 2: 'COATB_BPI22/32-83', 3: 'COATB_BPM13/24-72', 4: 'COATB_BPZJ2/1-49', 5: 'Q9T0Q9_BPFD/1-49', 6: 'COATB_BPIF1/22-73'}
    >>> AlignIO.write([alignment], "PF05371_seed.phy", "phylip")

This code used a Python dictionary to record a simple mapping from the new sequence system to the original identifier:

    {
        0: "COATB_BPIKE/30-81",
        1: "Q9T0Q8_BPIKE/1-52",
        2: "COATB_BPI22/32-83",
        # ...
    }

Here is the new (strict) PHYLIP format output:

     7 52
    seq0       AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
    seq1       AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
    seq2       DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
    seq3       AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    seq4       AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
    seq5       AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
    seq6       FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS

               KA
               RA
               KA
               KA
               KA
               KA
               RA

In general, because of the identifier limitation, working with strict PHYLIP file formats shouldn’t be your first choice. Using the PFAM/Stockholm format on the other hand allows you to record a lot of additional annotation too.

### Getting your alignment objects as formatted strings

The `Bio.AlignIO` interface is based on handles, which means if you want to get your alignment(s) into a string in a particular file format you need to do a little bit more work (see below). However, you will probably prefer to call Python’s built-in `format` function on the alignment object. This takes an output format specification as a single argument, a lower case string which is supported by `Bio.AlignIO` as an output format. For example:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> print(format(alignment, "clustal"))
    CLUSTAL X (1.81) multiple sequence alignment

    COATB_BPIKE/30-81                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSS
    Q9T0Q8_BPIKE/1-52                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVS
    COATB_BPI22/32-83                   DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSS
    ...

Without an output format specification, `format` returns the same output as `str`.

As described in Section The format method, the `SeqRecord` object has a similar method using output formats supported by `Bio.SeqIO`.

Internally `format` is calling `Bio.AlignIO.write()` with a `StringIO` handle. You can do this in your own code if for example you are using an older version of Biopython:

    >>> from io import StringIO
    >>> from Bio import AlignIO
    >>> alignments = AlignIO.parse("PF05371_seed.sth", "stockholm")
    >>> out_handle = StringIO()
    >>> AlignIO.write(alignments, out_handle, "clustal")
    1
    >>> clustal_data = out_handle.getvalue()
    >>> print(clustal_data)
    CLUSTAL X (1.81) multiple sequence alignment

    COATB_BPIKE/30-81                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSS
    Q9T0Q8_BPIKE/1-52                   AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVS
    COATB_BPI22/32-83                   DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSS
    COATB_BPM13/24-72                   AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTS
    ...

## Manipulating Alignments

Now that we’ve covered loading and saving alignments, we’ll look at what else you can do with them.

### Slicing alignments

First of all, in some senses the alignment objects act like a Python `list` of `SeqRecord` objects (the rows). With this model in mind hopefully the actions of `len()` (the number of rows) and iteration (each row as a `SeqRecord`) make sense:

    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> print("Number of rows: %i" % len(alignment))
    Number of rows: 7
    >>> for record in alignment:
    ...     print("%s - %s" % (record.seq, record.id))
    ...
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA - COATB_BPIKE/30-81
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA - Q9T0Q8_BPIKE/1-52
    DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA - COATB_BPI22/32-83
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - COATB_BPM13/24-72
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA - COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - Q9T0Q9_BPFD/1-49
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA - COATB_BPIF1/22-73

You can also use the list-like `append` and `extend` methods to add more rows to the alignment (as `SeqRecord` objects). Keeping the list metaphor in mind, simple slicing of the alignment should also make sense - it selects some of the rows giving back another alignment object:

    >>> print(alignment)
    Alignment with 7 rows and 52 columns
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRL...SKA COATB_BPIKE/30-81
    AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKL...SRA Q9T0Q8_BPIKE/1-52
    DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRL...SKA COATB_BPI22/32-83
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73
    >>> print(alignment[3:7])
    Alignment with 4 rows and 52 columns
    AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49
    AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49
    FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73

What if you wanted to select by column? Those of you who have used the NumPy matrix or array objects won’t be surprised at this - you use a double index.

    >>> print(alignment[2, 6])
    T

Using two integer indices pulls out a single letter, short hand for this:

    >>> print(alignment[2].seq[6])
    T

You can pull out a single column as a string like this:

    >>> print(alignment[:, 6])
    TTT---T

You can also select a range of columns. For example, to pick out those same three rows we extracted earlier, but take just their first six columns:

    >>> print(alignment[3:6, :6])
    Alignment with 3 rows and 6 columns
    AEGDDP COATB_BPM13/24-72
    AEGDDP COATB_BPZJ2/1-49
    AEGDDP Q9T0Q9_BPFD/1-49

Leaving the first index as `:` means take all the rows:

    >>> print(alignment[:, :6])
    Alignment with 7 rows and 6 columns
    AEPNAA COATB_BPIKE/30-81
    AEPNAA Q9T0Q8_BPIKE/1-52
    DGTSTA COATB_BPI22/32-83
    AEGDDP COATB_BPM13/24-72
    AEGDDP COATB_BPZJ2/1-49
    AEGDDP Q9T0Q9_BPFD/1-49
    FAADDA COATB_BPIF1/22-73

This brings us to a neat way to remove a section. Notice columns 7, 8 and 9 which are gaps in three of the seven sequences:

    >>> print(alignment[:, 6:9])
    Alignment with 7 rows and 3 columns
    TNY COATB_BPIKE/30-81
    TNY Q9T0Q8_BPIKE/1-52
    TSY COATB_BPI22/32-83
    --- COATB_BPM13/24-72
    --- COATB_BPZJ2/1-49
    --- Q9T0Q9_BPFD/1-49
    TSQ COATB_BPIF1/22-73

Again, you can slice to get everything after the ninth column:

    >>> print(alignment[:, 9:])
    Alignment with 7 rows and 43 columns
    ATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81
    ATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52
    ATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83
    AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72
    AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49
    AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49
    AKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73

Now, the interesting thing is that addition of alignment objects works by column. This lets you do this as a way to remove a block of columns:

    >>> edited = alignment[:, :6] + alignment[:, 9:]
    >>> print(edited)
    Alignment with 7 rows and 49 columns
    AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81
    AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52
    DGTSTAATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83
    AEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72
    AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49
    AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49
    FAADDAAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73

Another common use of alignment addition would be to combine alignments for several different genes into a meta-alignment. Watch out though - the identifiers need to match up (see Section Adding SeqRecord objects for how adding `SeqRecord` objects works). You may find it helpful to first sort the alignment rows alphabetically by id:

    >>> edited.sort()
    >>> print(edited)
    Alignment with 7 rows and 49 columns
    DGTSTAATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83
    FAADDAAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73
    AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81
    AEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72
    AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49
    AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52
    AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49

Note that you can only add two alignments together if they have the same number of rows.

### Alignments as arrays

Depending on what you are doing, it can be more useful to turn the alignment object into an array of letters – and you can do this with NumPy:

    >>> import numpy as np
    >>> from Bio import AlignIO
    >>> alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
    >>> align_array = np.array(alignment)
    >>> print("Array shape %i by %i" % align_array.shape)
    Array shape 7 by 52
    >>> align_array[:, :10]
    array([['A', 'E', 'P', 'N', 'A', 'A', 'T', 'N', 'Y', 'A'],
           ['A', 'E', 'P', 'N', 'A', 'A', 'T', 'N', 'Y', 'A'],
           ['D', 'G', 'T', 'S', 'T', 'A', 'T', 'S', 'Y', 'A'],
           ['A', 'E', 'G', 'D', 'D', 'P', '-', '-', '-', 'A'],
           ['A', 'E', 'G', 'D', 'D', 'P', '-', '-', '-', 'A'],
           ['A', 'E', 'G', 'D', 'D', 'P', '-', '-', '-', 'A'],
           ['F', 'A', 'A', 'D', 'D', 'A', 'T', 'S', 'Q', 'A']],...

Note that this leaves the original Biopython alignment object and the NumPy array in memory as separate objects - editing one will not update the other!

### Counting substitutions

The `substitutions` property of an alignment reports how often letters in the alignment are substituted for each other. This is calculated by taking all pairs of rows in the alignment, counting the number of times two letters are aligned to each other, and summing this over all pairs. For example,

    >>> from Bio.Seq import Seq
    >>> from Bio.SeqRecord import SeqRecord
    >>> from Bio.Align import MultipleSeqAlignment
    >>> msa = MultipleSeqAlignment(
    ...     [
    ...         SeqRecord(Seq("ACTCCTA"), id="seq1"),
    ...         SeqRecord(Seq("AAT-CTA"), id="seq2"),
    ...         SeqRecord(Seq("CCTACT-"), id="seq3"),
    ...         SeqRecord(Seq("TCTCCTC"), id="seq4"),
    ...     ]
    ... )
    >>> print(msa)
    Alignment with 4 rows and 7 columns
    ACTCCTA seq1
    AAT-CTA seq2
    CCTACT- seq3
    TCTCCTC seq4
    >>> substitutions = msa.substitutions
    >>> print(substitutions)
        A    C    T
    A 2.0  4.5  1.0
    C 4.5 10.0  0.5
    T 1.0  0.5 12.0

As the ordering of pairs is arbitrary, counts are divided equally above and below the diagonal. For example, the 9 alignments of `A` to `C` are stored as 4.5 at position `['A', 'C']` and 4.5 at position `['C', 'A']`. This arrangement helps to make the math easier when calculating a substitution matrix from these counts, as described in Section Substitution matrices.

Note that `msa.substitutions` contains entries for the letters appearing in the alignment only. You can use the `select` method to add entries for missing letters, for example

    >>> m = substitutions.select("ATCG")
    >>> print(m)
        A    T    C   G
    A 2.0  1.0  4.5 0.0
    T 1.0 12.0  0.5 0.0
    C 4.5  0.5 10.0 0.0
    G 0.0  0.0  0.0 0.0

This also allows you to change the order of letters in the alphabet.

## Getting a new-style Alignment object

Use the `alignment` property to create a new-style `Alignment` object (see section Alignment objects) from an old-style `MultipleSeqAlignment` object:

    >>> type(msa)
    <class 'Bio.Align.MultipleSeqAlignment'>
    >>> print(msa)
    Alignment with 4 rows and 7 columns
    ACTCCTA seq1
    AAT-CTA seq2
    CCTACT- seq3
    TCTCCTC seq4
    >>> alignment = msa.alignment
    >>> type(alignment)
    <class 'Bio.Align.Alignment'>
    >>> print(alignment)
    seq1              0 ACTCCTA 7
    seq2              0 AAT-CTA 6
    seq3              0 CCTACT- 6
    seq4              0 TCTCCTC 7

Note that the `alignment` property creates and returns a new `Alignment` object that is consistent with the information stored in the `MultipleSeqAlignment` object at the time the `Alignment` object is created. Any changes to the `MultipleSeqAlignment` after calling the `alignment` property will not propagate to the `Alignment` object. However, you can of course call the `alignment` property again to create a new `Alignment` object consistent with the updated `MultipleSeqAlignment` object.

## Calculating a substitution matrix from a multiple sequence alignment

You can create your own substitution matrix from an alignment. In this example, we’ll first read a protein sequence alignment from the Clustalw file protein.aln (also available online here)

    >>> from Bio import AlignIO
    >>> filename = "protein.aln"
    >>> msa = AlignIO.read(filename, "clustal")

Section ClustalW contains more information on doing this.

The `substitutions` property of the alignment stores the number of times different residues substitute for each other:

    >>> observed_frequencies = msa.substitutions

To make the example more readable, we’ll select only amino acids with polar charged side chains:

    >>> observed_frequencies = observed_frequencies.select("DEHKR")
    >>> print(observed_frequencies)
           D      E      H      K      R
    D 2360.0  255.5    7.5    0.5   25.0
    E  255.5 3305.0   16.5   27.0    2.0
    H    7.5   16.5 1235.0   16.0    8.5
    K    0.5   27.0   16.0 3218.0  116.5
    R   25.0    2.0    8.5  116.5 2079.0

Rows and columns for other amino acids were removed from the matrix.

Next, we normalize the matrix:

    >>> import numpy as np
    >>> observed_frequencies /= np.sum(observed_frequencies)

Summing over rows or columns gives the relative frequency of occurrence of each residue:

    >>> residue_frequencies = np.sum(observed_frequencies, 0)
    >>> print(residue_frequencies.format("%.4f"))
    D 0.2015
    E 0.2743
    H 0.0976
    K 0.2569
    R 0.1697

    >>> sum(residue_frequencies) == 1.0
    True

The expected frequency of residue pairs is then

    >>> expected_frequencies = np.dot(
    ...     residue_frequencies[:, None], residue_frequencies[None, :]
    ... )
    >>> print(expected_frequencies.format("%.4f"))
           D      E      H      K      R
    D 0.0406 0.0553 0.0197 0.0518 0.0342
    E 0.0553 0.0752 0.0268 0.0705 0.0465
    H 0.0197 0.0268 0.0095 0.0251 0.0166
    K 0.0518 0.0705 0.0251 0.0660 0.0436
    R 0.0342 0.0465 0.0166 0.0436 0.0288

Here, `residue_frequencies[:, None]` creates a 2D array consisting of a single column with the values of `residue_frequencies`, and `residue_frequencies[None, :]` a 2D array with these values as a single row. Taking their dot product (inner product) creates a matrix of expected frequencies where each entry consists of two `residue_frequencies` values multiplied with each other. For example, `expected_frequencies['D', 'E']` is equal to `residue_frequencies['D'] * residue_frequencies['E']`.

We can now calculate the log-odds matrix by dividing the observed frequencies by the expected frequencies and taking the logarithm:

    >>> m = np.log2(observed_frequencies / expected_frequencies)
    >>> print(m)
          D    E    H     K    R
    D   2.1 -1.5 -5.1 -10.4 -4.2
    E  -1.5  1.7 -4.4  -5.1 -8.3
    H  -5.1 -4.4  3.3  -4.4 -4.7
    K -10.4 -5.1 -4.4   1.9 -2.3
    R  -4.2 -8.3 -4.7  -2.3  2.5

This matrix can be used as the substitution matrix when performing alignments. For example,

    >>> from Bio.Align import PairwiseAligner
    >>> aligner = PairwiseAligner()
    >>> aligner.substitution_matrix = m
    >>> aligner.gap_score = -3.0
    >>> alignments = aligner.align("DEHEK", "DHHKK")
    >>> print(alignments[0])
    target            0 DEHEK 5
                      0 |.|.| 5
    query             0 DHHKK 5

    >>> print("%.2f" % alignments.score)
    -2.18
    >>> score = m["D", "D"] + m["E", "H"] + m["H", "H"] + m["E", "K"] + m["K", "K"]
    >>> print("%.2f" % score)
    -2.18

## Alignment Tools

There are lots of algorithms out there for aligning sequences, both pairwise alignments and multiple sequence alignments. These calculations are relatively slow, and you generally wouldn’t want to write such an algorithm in Python. For pairwise alignments, you can use Biopython’s `PairwiseAligner` (see Chapter Pairwise sequence alignment), which is implemented in C and therefore fast. Alternatively, you can run an external alignment program by invoking it from Python. Normally you would:

1. Prepare an input file of your unaligned sequences, typically this will be a FASTA file which you might create using `Bio.SeqIO` (see Chapter Sequence Input/Output).

2. Run the alignment program by running its command using Python’s `subprocess` module.

3. Read the output from the tool, i.e. your aligned sequences, typically using `Bio.AlignIO` (see earlier in this chapter).

Here, we will show a few examples of this workflow.

### ClustalW

ClustalW is a popular command line tool for multiple sequence alignment (there is also a graphical interface called ClustalX). Before trying to use ClustalW from within Python, you should first try running the ClustalW tool yourself by hand at the command line, to familiarize yourself the other options.

For the most basic usage, all you need is to have a FASTA input file, such as opuntia.fasta (available online or in the Doc/examples subdirectory of the Biopython source code). This is a small FASTA file containing seven prickly-pear DNA sequences (from the cactus family Opuntia). By default ClustalW will generate an alignment and guide tree file with names based on the input FASTA file, in this case `opuntia.aln` and `opuntia.dnd`, but you can override this or make it explicit:

    >>> import subprocess
    >>> cmd = "clustalw2 -infile=opuntia.fasta"
    >>> results = subprocess.run(cmd, shell=True, stdout=subprocess.PIPE, text=True)

Notice here we have given the executable name as `clustalw2`, indicating we have version two installed, which has a different filename to version one (`clustalw`, the default). Fortunately both versions support the same set of arguments at the command line (and indeed, should be functionally identical).

You may find that even though you have ClustalW installed, the above command doesn’t work – you may get a message about “command not found” (especially on Windows). This indicated that the ClustalW executable is not on your PATH (an environment variable, a list of directories to be searched). You can either update your PATH setting to include the location of your copy of ClustalW tools (how you do this will depend on your OS), or simply type in the full path of the tool. Remember, in Python strings `\n` and `\t` are by default interpreted as a new line and a tab – which is why we’re put a letter “r” at the start for a raw string that isn’t translated in this way. This is generally good practice when specifying a Windows style file name.

    >>> import os
    >>> clustalw_exe = r"C:\Program Files\new clustal\clustalw2.exe"
    >>> assert os.path.isfile(clustalw_exe), "Clustal W executable missing"
    >>> cmd = clustalw_exe + " -infile=opuntia.fasta"
    >>> results = subprocess.run(cmd, shell=True, stdout=subprocess.PIPE, text=True)

Now, at this point it helps to know about how command line tools “work”. When you run a tool at the command line, it will often print text output directly to screen. This text can be captured or redirected, via two “pipes”, called standard output (the normal results) and standard error (for error messages and debug messages). There is also standard input, which is any text fed into the tool. These names get shortened to stdin, stdout and stderr. When the tool finishes, it has a return code (an integer), which by convention is zero for success, while a non-zero return code indicates that an error has occurred.

In the example of ClustalW above, when run at the command line all the important output is written directly to the output files. Everything normally printed to screen while you wait is captured in `results.stdout` and `results.stderr`, while the return code is stored in `results.returncode`.

What we care about are the two output files, the alignment and the guide tree. We didn’t tell ClustalW what filenames to use, but it defaults to picking names based on the input file. In this case the output should be in the file `opuntia.aln`. You should be able to work out how to read in the alignment using `Bio.AlignIO` by now:

    >>> from Bio import AlignIO
    >>> align = AlignIO.read("opuntia.aln", "clustal")
    >>> print(align)
    Alignment with 7 rows and 906 columns
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF191
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF191
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF191
    TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF191
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF191
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF191
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273291|gb|AF191665.1|AF191

In case you are interested (and this is an aside from the main thrust of this chapter), the `opuntia.dnd` file ClustalW creates is just a standard Newick tree file, and `Bio.Phylo` can parse these:

    >>> from Bio import Phylo
    >>> tree = Phylo.read("opuntia.dnd", "newick")
    >>> Phylo.draw_ascii(tree)
                                 _______________ gi|6273291|gb|AF191665.1|AF191665
      __________________________|
     |                          |   ______ gi|6273290|gb|AF191664.1|AF191664
     |                          |__|
     |                             |_____ gi|6273289|gb|AF191663.1|AF191663
     |
    _|_________________ gi|6273287|gb|AF191661.1|AF191661
     |
     |__________ gi|6273286|gb|AF191660.1|AF191660
     |
     |    __ gi|6273285|gb|AF191659.1|AF191659
     |___|
         | gi|6273284|gb|AF191658.1|AF191658

Chapter Phylogenetics with Bio.Phylo covers Biopython’s support for phylogenetic trees in more depth.

### MUSCLE

MUSCLE is a more recent multiple sequence alignment tool than ClustalW. As before, we recommend you try using MUSCLE from the command line before trying to run it from Python.

For the most basic usage, all you need is to have a FASTA input file, such as opuntia.fasta (available online or in the Doc/examples subdirectory of the Biopython source code). You can then tell MUSCLE to read in this FASTA file, and write the alignment to an output file named `opuntia.txt`:

    >>> import subprocess
    >>> cmd = "muscle -align opuntia.fasta -output opuntia.txt"
    >>> results = subprocess.run(cmd, shell=True, stdout=subprocess.PIPE, text=True)

MUSCLE will output the alignment as a FASTA file (using gapped sequences). The `Bio.AlignIO` module is able to read this alignment using `format="fasta"`:

    >>> from Bio import AlignIO
    >>> align = AlignIO.read("opuntia.txt", "fasta")
    >>> print(align)
    Alignment with 7 rows and 906 columns
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF191663
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273291|gb|AF191665.1|AF191665
    TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF191664
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF191661
    TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF191660
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF191659
    TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF191658

You can also set the other optional parameters; see MUSCLE’s built-in help for details.

### EMBOSS needle and water

The EMBOSS suite includes the `water` and `needle` tools for Smith-Waterman algorithm local alignment, and Needleman-Wunsch global alignment. The tools share the same style interface, so switching between the two is trivial – we’ll just use `needle` here.

Suppose you want to do a global pairwise alignment between two sequences, prepared in FASTA format as follows:

    >HBA_HUMAN
    MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHG
    KKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTP
    AVHASLDKFLASVSTVLTSKYR

in a file `alpha.faa`, and secondly in a file `beta.faa`:

    >HBB_HUMAN
    MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK
    VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG
    KEFTPPVQAAYQKVVAGVANALAHKYH

You can find copies of these example files with the Biopython source code under the `Doc/examples/` directory.

The command to align these two sequences against each other using `needle` is as follows:

    needle -outfile=needle.txt -asequence=alpha.faa -bsequence=beta.faa -gapopen=10 -gapextend=0.5

Why not try running this by hand at the command prompt? You should see it does a pairwise comparison and records the output in the file `needle.txt` (in the default EMBOSS alignment file format).

Even if you have EMBOSS installed, running this command may not work – you might get a message about “command not found” (especially on Windows). This probably means that the EMBOSS tools are not on your PATH environment variable. You can either update your PATH setting, or simply use the full path to the tool, for example:

    C:\EMBOSS\needle.exe -outfile=needle.txt -asequence=alpha.faa -bsequence=beta.faa -gapopen=10 -gapextend=0.5

Next we want to use Python to run this command for us. As explained above, for full control, we recommend you use Python’s built-in `subprocess` module:

    >>> import sys
    >>> import subprocess
    >>> cmd = "needle -outfile=needle.txt -asequence=alpha.faa -bsequence=beta.faa -gapopen=10 -gapextend=0.5"
    >>> results = subprocess.run(
    ...     cmd,
    ...     stdout=subprocess.PIPE,
    ...     stderr=subprocess.PIPE,
    ...     text=True,
    ...     shell=(sys, platform != "win32"),
    ... )
    >>> print(results.stdout)

    >>> print(results.stderr)
    Needleman-Wunsch global alignment of two sequences

Next we can load the output file with `Bio.AlignIO` as discussed earlier in this chapter, as the `emboss` format:

    >>> from Bio import AlignIO
    >>> align = AlignIO.read("needle.txt", "emboss")
    >>> print(align)
    Alignment with 2 rows and 149 columns
    MV-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTY...KYR HBA_HUMAN
    MVHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRF...KYH HBB_HUMAN

In this example, we told EMBOSS to write the output to a file, but you can tell it to write the output to stdout instead (useful if you don’t want a temporary output file to get rid of – use `outfile=stdout` argument):

    >>> cmd = "needle -outfile=stdout -asequence=alpha.faa -bsequence=beta.faa -gapopen=10 -gapextend=0.5"
    >>> child = subprocess.Popen(
    ...     cmd,
    ...     stdout=subprocess.PIPE,
    ...     stderr=subprocess.PIPE,
    ...     text=True,
    ...     shell=(sys.platform != "win32"),
    ... )
    >>> align = AlignIO.read(child.stdout, "emboss")
    >>> print(align)
    Alignment with 2 rows and 149 columns
    MV-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTY...KYR HBA_HUMAN
    MVHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRF...KYH HBB_HUMAN

Similarly, it is possible to read one of the inputs from stdin (e.g. `asequence="stdin"`).

This has only scratched the surface of what you can do with `needle` and `water`. One useful trick is that the second file can contain multiple sequences (say five), and then EMBOSS will do five pairwise alignments.

Biopython v: 1.86

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