\chapter{Multiple Sequence Alignment objects}

\label{chapter:Bio.AlignIO}

This chapter is about Multiple Sequence Alignments, by which 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 \verb|SeqRecord| object internally.

We will introduce the \verb|MultipleSeqAlignment| object which holds this kind of data,

and the \verb|Bio.AlignIO| module for reading and writing them as various file formats

(following the design of the \verb|Bio.SeqIO| module from the previous chapter).

Note that both \verb|Bio.SeqIO| and \verb|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 our command line wrappers for common multiple

sequence alignment tools like ClustalW and MUSCLE.

\section{Parsing or Reading Sequence Alignments}

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

Using \verb|Bio.AlignIO.parse()| will return an {\it iterator} which gives \verb|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 \verb|seqboot|, or multiple pairwise alignments from the EMBOSS tools \verb|water| or \verb|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 \verb|Bio.AlignIO.read()| function which returns a single \verb|MultipleSeqAlignment| object.

Both functions expect two mandatory arguments:

\begin{enumerate}

\item The first argument is a {\it handle} to read the data from, typically an open file (see Section~\ref{sec:appendix-handles}), or a filename.

\item The second argument is a lower case string specifying the alignment format. As in \verb|Bio.SeqIO| we don't try and guess the file format for you! See \url{http://biopython.org/wiki/AlignIO} for a full listing of supported formats.

\end{enumerate}

\noindent There is also an optional \verb|seq_count| argument which is discussed in Section~\ref{sec:AlignIO-count-argument} below for dealing with ambiguous file formats which may contain more than one alignment.

A further optional \verb|alphabet| argument allowing you to specify the expected alphabet. This can be useful as many alignment file formats do not explicitly label the sequences as RNA, DNA or protein -- which means \verb|Bio.AlignIO| will default to using a generic alphabet.

\subsection{Single Alignments}

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

\begin{verbatim}

# 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

//

\end{verbatim}

This is the seed alignment for the Phage\_Coat\_Gp8 (PF05371) PFAM entry, downloaded from a now out of date release of PFAM from \url{http://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):

%doctest examples

\begin{verbatim}

>>> from Bio import AlignIO

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

\end{verbatim}

\noindent This code will print out a summary of the alignment:

%cont-doctest

\begin{verbatim}

>>> print(alignment)

SingleLetterAlphabet() 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

\end{verbatim}

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 \verb|SeqRecord| objects:

%doctest examples

\begin{verbatim}

>>> 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

\end{verbatim}

You could also use the alignment object's \verb|format| method to show it in a particular file format -- see Section~\ref{sec:alignment-format-method} 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:

%cont-doctest

\begin{verbatim}

>>> 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;']

\end{verbatim}

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

\begin{verbatim}

>>> for record in alignment:

... print(record)

...

\end{verbatim}

Sanger provide a nice web interface at \url{http://pfam.sanger.ac.uk/family?acc=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:

\begin{verbatim}

>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

\end{verbatim}

\noindent 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:

\begin{verbatim}

from Bio import AlignIO

alignment = AlignIO.read("PF05371_seed.faa", "fasta")

print(alignment)

\end{verbatim}

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 \verb|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 \verb|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 (\url{http://biopython.org/wiki/AlignIO}) and in the built in documentation (also \href{http://biopython.org/DIST/docs/api/Bio.AlignIO-module.html}{online}):

\begin{verbatim}

>>> from Bio import AlignIO

>>> help(AlignIO)

...

\end{verbatim}

\subsection{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 \verb|Bio.AlignIO.parse()| function.

Suppose you have a small alignment in PHYLIP format:

\begin{verbatim}

5 6

Alpha AACAAC

Beta AACCCC

Gamma ACCAAC

Delta CCACCA

Epsilon CCAAAC

\end{verbatim}

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 \verb|bootseq|. This would give output something like this, which has been abbreviated for conciseness:

\begin{verbatim}

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

\end{verbatim}

If you wanted to read this in using \verb|Bio.AlignIO| you could use:

%TODO - Replace the print blank line with print()?

\begin{verbatim}

from Bio import AlignIO

alignments = AlignIO.parse("resampled.phy", "phylip")

for alignment in alignments:

print(alignment)

print("")

\end{verbatim}

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

\begin{verbatim}

SingleLetterAlphabet() alignment with 5 rows and 6 columns

AAACCA Alpha

AAACCC Beta

ACCCCA Gamma

CCCAAC Delta

CCCAAA Epsilon

SingleLetterAlphabet() alignment with 5 rows and 6 columns

AAACAA Alpha

AAACCC Beta

ACCCAA Gamma

CCCACC Delta

CCCAAA Epsilon

SingleLetterAlphabet() alignment with 5 rows and 6 columns

AAAAAC Alpha

AAACCC Beta

AACAAC Gamma

CCCCCA Delta

CCCAAC Epsilon

...

SingleLetterAlphabet() alignment with 5 rows and 6 columns

AAAACC Alpha

ACCCCC Beta

AAAACC Gamma

CCCCAA Delta

CAAACC Epsilon

\end{verbatim}

As with the function \verb|Bio.SeqIO.parse()|, using \verb|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:

\begin{verbatim}

from Bio import AlignIO

alignments = list(AlignIO.parse("resampled.phy", "phylip"))

last_align = alignments[-1]

first_align = alignments[0]

\end{verbatim}

\subsection{Ambiguous Alignments}

\label{sec:AlignIO-count-argument}

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:

\begin{verbatim}

>Alpha

ACTACGACTAGCTCAG--G

>Beta

ACTACCGCTAGCTCAGAAG

>Gamma

ACTACGGCTAGCACAGAAG

>Alpha

ACTACGACTAGCTCAGG--

>Beta

ACTACCGCTAGCTCAGAAG

>Gamma

ACTACGGCTAGCACAGAAG

\end{verbatim}

\noindent 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?

\begin{verbatim}

>Alpha

ACTACGACTAGCTCAG--G

>Beta

ACTACCGCTAGCTCAGAAG

>Alpha

ACTACGACTAGCTCAGG--

>Gamma

ACTACGGCTAGCACAGAAG

>Alpha

ACTACGACTAGCTCAGG--

>Delta

ACTACGGCTAGCACAGAAG

\end{verbatim}

\noindent 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:

\begin{verbatim}

>Alpha

ACTACGACTAGCTCAG--G

>XXX

ACTACCGCTAGCTCAGAAG

>Alpha

ACTACGACTAGCTCAGG

>YYY

ACTACGGCAAGCACAGG

>Alpha

--ACTACGAC--TAGCTCAGG

>ZZZ

GGACTACGACAATAGCTCAGG

\end{verbatim}

\noindent 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 \verb|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 \verb|needle| and \verb|water| -- although in this situation, \verb|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 \verb|Bio.AlignIO.parse()| with the optional \verb|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:

%TODO - Replace the print blank line with print()?

\begin{verbatim}

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("")

\end{verbatim}

\noindent giving:

\begin{verbatim}

Alignment length 19

ACTACGACTAGCTCAG--G - Alpha

ACTACCGCTAGCTCAGAAG - XXX

Alignment length 17

ACTACGACTAGCTCAGG - Alpha

ACTACGGCAAGCACAGG - YYY

Alignment length 21

--ACTACGAC--TAGCTCAGG - Alpha

GGACTACGACAATAGCTCAGG - ZZZ

\end{verbatim}

Using \verb|Bio.AlignIO.read()| or \verb|Bio.AlignIO.parse()| without the \verb|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 \verb|Bio.AlignIO| to determine the number of sequences in each alignment directly, then the \verb|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 \verb|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 \verb|Bio.AlignIO|. In this case, you could consider reading in the sequences themselves using \verb|Bio.SeqIO| and batching them together to create the alignments as appropriate.

\section{Writing Alignments}

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

Here is an example, where we start by creating a few \verb|MultipleSeqAlignment| objects the hard way (by hand, rather than by loading them from a file).

Note we create some \verb|SeqRecord| objects to construct the alignment from.

\begin{verbatim}

from Bio.Alphabet import generic_dna

from Bio.Seq import Seq

from Bio.SeqRecord import SeqRecord

from Bio.Align import MultipleSeqAlignment

align1 = MultipleSeqAlignment([

SeqRecord(Seq("ACTGCTAGCTAG", generic_dna), id="Alpha"),

SeqRecord(Seq("ACT-CTAGCTAG", generic_dna), id="Beta"),

SeqRecord(Seq("ACTGCTAGDTAG", generic_dna), id="Gamma"),

])

align2 = MultipleSeqAlignment([

SeqRecord(Seq("GTCAGC-AG", generic_dna), id="Delta"),

SeqRecord(Seq("GACAGCTAG", generic_dna), id="Epsilon"),

SeqRecord(Seq("GTCAGCTAG", generic_dna), id="Zeta"),

])

align3 = MultipleSeqAlignment([

SeqRecord(Seq("ACTAGTACAGCTG", generic_dna), id="Eta"),

SeqRecord(Seq("ACTAGTACAGCT-", generic_dna), id="Theta"),

SeqRecord(Seq("-CTACTACAGGTG", generic_dna), id="Iota"),

])

my_alignments = [align1, align2, align3]

\end{verbatim}

\noindent Now we have a list of \verb|Alignment| objects, we'll write them to a PHYLIP format file:

\begin{verbatim}

from Bio import AlignIO

AlignIO.write(my_alignments, "my_example.phy", "phylip")

\end{verbatim}

\noindent And if you open this file in your favourite text editor it should look like this:

\begin{verbatim}

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

\end{verbatim}

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

\emph{Note} - If you tell the \verb|Bio.AlignIO.write()| function to write to a file that already exists, the old file will be overwritten without any warning.

\subsection{Converting between sequence alignment file formats}

\label{sec:converting-alignments}

Converting between sequence alignment file formats with \verb|Bio.AlignIO| works

in the same way as converting between sequence file formats with \verb|Bio.SeqIO|

(Section~\ref{sec:SeqIO-conversion}). We load generally the alignment(s) using

\verb|Bio.AlignIO.parse()| and then save them using the \verb|Bio.AlignIO.write()|

-- or just use the \verb|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:

\begin{verbatim}

from Bio import AlignIO

count = AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.aln", "clustal")

print("Converted %i alignments" % count)

\end{verbatim}

Or, using \verb|Bio.AlignIO.parse()| and \verb|Bio.AlignIO.write()|:

\begin{verbatim}

from Bio import AlignIO

alignments = AlignIO.parse("PF05371_seed.sth", "stockholm")

count = AlignIO.write(alignments, "PF05371_seed.aln", "clustal")

print("Converted %i alignments" % count)

\end{verbatim}

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

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

\begin{verbatim}

from Bio import AlignIO

alignment = AlignIO.read("PF05371_seed.sth", "stockholm")

AlignIO.write([alignment], "PF05371_seed.aln", "clustal")

\end{verbatim}

Either way, you should end up with the same new Clustal W format file ``PF05371\_seed.aln'' with the following content:

\begin{verbatim}

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

\end{verbatim}

Alternatively, you could make a PHYLIP format file which we'll name ``PF05371\_seed.phy'':

\begin{verbatim}

from Bio import AlignIO

AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip")

\end{verbatim}

This time the output looks like this:

\begin{verbatim}

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

\end{verbatim}

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:

\begin{verbatim}

from Bio import AlignIO

AlignIO.convert("PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip-relaxed")

\end{verbatim}

This time the output looks like this, using a longer indentation to

allow all the identifers to be given in full::

\begin{verbatim}

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

\end{verbatim}

If you have to work with the original strict PHYLIP format, then you may need to

compress the identifers somehow -- or assign your own names or numbering system.

This following bit of code manipulates the record identifiers before saving the output:

\begin{verbatim}

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)

AlignIO.write([alignment], "PF05371_seed.phy", "phylip")

\end{verbatim}

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

\begin{verbatim}

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

\end{verbatim}

\noindent Here is the new (strict) PHYLIP format output:

\begin{verbatim}

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

\end{verbatim}

\noindent In general, because of the identifier limitation, working with

\textit{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.

\subsection{Getting your alignment objects as formatted strings}

\label{sec:alignment-format-method}

The \verb|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 take advantage of the alignment object's \verb|format()| method.

This takes a single mandatory argument, a lower case string which is supported by \verb|Bio.AlignIO| as an output format. For example:

\begin{verbatim}

from Bio import AlignIO

alignment = AlignIO.read("PF05371_seed.sth", "stockholm")

print(alignment.format("clustal"))

\end{verbatim}

As described in Section~\ref{sec:SeqRecord-format}, the \verb|SeqRecord| object has a similar method using output formats supported by \verb|Bio.SeqIO|.

Internally the \verb|format()| method is using the \verb|StringIO| string based handle and calling

\verb|Bio.AlignIO.write()|. You can do this in your own code if for example you are using an

older version of Biopython:

\begin{verbatim}

from Bio import AlignIO

from StringIO import StringIO

alignments = AlignIO.parse("PF05371_seed.sth", "stockholm")

out_handle = StringIO()

AlignIO.write(alignments, out_handle, "clustal")

clustal_data = out_handle.getvalue()

print(clustal_data)

\end{verbatim}

\section{Manipulating Alignments}

\label{sec:manipulating-alignments}

Now that we've covered loading and saving alignments, we'll look at what else you can do

with them.

\subsection{Slicing alignments}

First of all, in some senses the alignment objects act like a Python \verb|list| of

\verb|SeqRecord| objects (the rows). With this model in mind hopefully the actions

of \verb|len()| (the number of rows) and iteration (each row as a \verb|SeqRecord|)

make sense:

%doctest examples

\begin{verbatim}

>>> 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

\end{verbatim}

You can also use the list-like \verb|append| and \verb|extend| methods to add

more rows to the alignment (as \verb|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:

%cont-doctest

\begin{verbatim}

>>> print(alignment)

SingleLetterAlphabet() 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])

SingleLetterAlphabet() 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

\end{verbatim}

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.

%cont-doctest

\begin{verbatim}

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

T

\end{verbatim}

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

%cont-doctest

\begin{verbatim}

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

T

\end{verbatim}

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

%cont-doctest

\begin{verbatim}

>>> print(alignment[:, 6])

TTT---T

\end{verbatim}

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:

%cont-doctest

\begin{verbatim}

>>> print(alignment[3:6, :6])

SingleLetterAlphabet() alignment with 3 rows and 6 columns

AEGDDP COATB_BPM13/24-72

AEGDDP COATB_BPZJ2/1-49

AEGDDP Q9T0Q9_BPFD/1-49

\end{verbatim}

Leaving the first index as \verb|:| means take all the rows:

%cont-doctest

\begin{verbatim}

>>> print(alignment[:, :6])

SingleLetterAlphabet() 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

\end{verbatim}

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:

%cont-doctest

\begin{verbatim}

>>> print(alignment[:, 6:9])

SingleLetterAlphabet() 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

\end{verbatim}

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

%cont-doctest

\begin{verbatim}

>>> print(alignment[:, 9:])

SingleLetterAlphabet() 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

\end{verbatim}

\noindent 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:

%cont-doctest

\begin{verbatim}

>>> edited = alignment[:, :6] + alignment[:, 9:]

>>> print(edited)

SingleLetterAlphabet() 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

\end{verbatim}

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~\ref{sec:SeqRecord-addition} for how adding

\verb|SeqRecord| objects works). You may find it helpful to first sort the

alignment rows alphabetically by id:

%cont-doctest

\begin{verbatim}

>>> edited.sort()

>>> print(edited)

SingleLetterAlphabet() 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

\end{verbatim}

\noindent Note that you can only add two alignments together if they

have the same number of rows.

\subsection{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:

%This example fails under PyPy 2.0, https://bugs.pypy.org/issue1546

%doctest examples lib:numpy

\begin{verbatim}

>>> import numpy as np

>>> from Bio import AlignIO

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

>>> align_array = np.array([list(rec) for rec in alignment], np.character)

>>> print("Array shape %i by %i" % align_array.shape)

Array shape 7 by 52

\end{verbatim}

If you will be working heavily with the columns, you can tell NumPy to store

the array by column (as in Fortran) rather then its default of by row (as in C):

\begin{verbatim}

>>> align_array = np.array([list(rec) for rec in alignment], np.character, order="F")

\end{verbatim}

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!

\section{Alignment Tools}

\label{sec:alignment-tools}

There are \emph{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 Biopython contains

the \verb|Bio.pairwise2| module (see Section~\ref{sec:pairwise2}), which is supplemented by

functions written in C for speed enhancements. In addition, you can use Biopython to invoke a

command line tool on your behalf. Normally you would:

\begin{enumerate}

\item Prepare an input file of your unaligned sequences, typically this will be a FASTA file

which you might create using \verb|Bio.SeqIO| (see Chapter~\ref{chapter:Bio.SeqIO}).

\item Call the command line tool to process this input file, typically via one of Biopython's

command line wrappers (which we'll discuss here).

\item Read the output from the tool, i.e. your aligned sequences, typically using

\verb|Bio.AlignIO| (see earlier in this chapter).

\end{enumerate}

All the command line wrappers we're going to talk about in this chapter follow the same style.

You create a command line object specifying the options (e.g. the input filename and the

output filename), then invoke this command line via a Python operating system call (e.g.

using the \texttt{subprocess} module).

Most of these wrappers are defined in the \verb|Bio.Align.Applications| module:

\begin{verbatim}

>>> import Bio.Align.Applications

>>> dir(Bio.Align.Applications)

...

['ClustalwCommandline', 'DialignCommandline', 'MafftCommandline', 'MuscleCommandline',

'PrankCommandline', 'ProbconsCommandline', 'TCoffeeCommandline' ...]

\end{verbatim}

\noindent (Ignore the entries starting with an underscore -- these have

special meaning in Python.)

The module \verb|Bio.Emboss.Applications| has wrappers for some of the

\href{http://emboss.sourceforge.net/}{EMBOSS suite}, including

\texttt{needle} and \texttt{water}, which are described below in

Section~\ref{seq:emboss-needle-water}, and wrappers for the EMBOSS

packaged versions of the PHYLIP tools (which EMBOSS refer to as one

of their EMBASSY packages - third party tools with an EMBOSS style

interface).

We won't explore all these alignment tools here in the section, just a

sample, but the same principles apply.

\subsection{ClustalW}

\label{sec:align_clustal}

ClustalW is a popular command line tool for multiple sequence alignment

(there is also a graphical interface called ClustalX). Biopython's

\verb|Bio.Align.Applications| module has a wrapper for this alignment tool

(and several others).

Before trying to use ClustalW from within Python, you should first try running

the ClustalW tool yourself by hand at the command line, to familiarise

yourself the other options. You'll find the Biopython wrapper is very

faithful to the actual command line API:

\begin{verbatim}

>>> from Bio.Align.Applications import ClustalwCommandline

>>> help(ClustalwCommandline)

...

\end{verbatim}

For the most basic usage, all you need is to have a FASTA input file, such as

\href{https://raw.githubusercontent.com/biopython/biopython/master/Doc/examples/opuntia.fasta}{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 \textit{Opuntia}).

By default ClustalW will generate an alignment and guide tree file with names

based on the input FASTA file, in this case \texttt{opuntia.aln} and

\texttt{opuntia.dnd}, but you can override this or make it explicit:

%doctest

\begin{verbatim}

>>> from Bio.Align.Applications import ClustalwCommandline

>>> cline = ClustalwCommandline("clustalw2", infile="opuntia.fasta")

>>> print(cline)

clustalw2 -infile=opuntia.fasta

\end{verbatim}

Notice here we have given the executable name as \texttt{clustalw2},

indicating we have version two installed, which has a different filename to

version one (\texttt{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. For example:

%doctest

\begin{verbatim}

>>> import os

>>> from Bio.Align.Applications import ClustalwCommandline

>>> clustalw_exe = r"C:\Program Files\new clustal\clustalw2.exe"

>>> clustalw_cline = ClustalwCommandline(clustalw_exe, infile="opuntia.fasta")

\end{verbatim}

%Don't run it in the doctest

\begin{verbatim}

>>> assert os.path.isfile(clustalw_exe), "Clustal W executable missing"

>>> stdout, stderr = clustalw_cline()

\end{verbatim}

\noindent Remember, in Python strings \verb|\n| and \verb|\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.

Internally this uses the

\verb|subprocess| module which is now the recommended way to run another

program in Python. This replaces older options like the \verb|os.system()|

and the \verb|os.popen*| functions.

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.

When you run the command line tool like this via the Biopython wrapper,

it will wait for it to finish, and check the return code. If this is

non zero (indicating an error), an exception is raised. The wrapper

then returns two strings, stdout and stderr.

In the case of ClustalW, 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 (via stdout or stderr) is boring and can be

ignored (assuming it worked).

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 \verb|opuntia.aln|.

You should be able to work out how to read in the alignment using

\verb|Bio.AlignIO| by now:

%doctest examples

\begin{verbatim}

>>> from Bio import AlignIO

>>> align = AlignIO.read("opuntia.aln", "clustal")

>>> print(align)

SingleLetterAlphabet() 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

\end{verbatim}

In case you are interested (and this is an aside from the main thrust of this

chapter), the \texttt{opuntia.dnd} file ClustalW creates is just a standard

Newick tree file, and \verb|Bio.Phylo| can parse these: