biopywork at uga

8/9/2019 biopywork at uga

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Sequences and alignmentsNCBI EUtils and BLAST

PhylogeneticsProtein structures

IOB Workshop: BiopythonA programming toolkit for bioinformatics

Eric Talevich

Institute of Bioinformatics, University of Georgia

Mar. 29, 2012

Eric Talevich IOB Workshop: Biopython
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Getting startedwith

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Installing Python

Biopython is a library for the Python programming language.

First, youll need these installed:

Python 2.7 from http://python.org. It may already beinstalled on your computer. (Version 2.6 is OK, too.)

IDLE, a simple Integrated DeveLopment Environment.Usually bundled with the Python distribution.

Now, start an interactive session in IDLE. 1

1On your own, check out IPython (http://ipython.scipy.org/). Its an

enhanced Python interpreter that feels somewhat likeR.Eric Talevich IOB Workshop: Biopython
http://python.org/http://ipython.scipy.org/http://ipython.scipy.org/http://ipython.scipy.org/http://python.org/http://find/


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Installing Python packages

Biopython is a Python package. There are a few standard ways toinstall Python packages:

From source: Download from PyPI 2, unpack and install with the

included setup.py script.easy install: Install from source 3, then use the easy install

command to fetch install all other packages by name:$ easy install

pip: Like easy install, use pip 4 to manage packages:$ pip install

2http://pypi.python.org/pypi/3http://pypi.python.org/pypi/setuptools

4http://pypi.python.org/pypi/pipEric Talevich IOB Workshop: Biopython

S d li
http://pypi.python.org/pypi/http://pypi.python.org/pypi/setuptoolshttp://pypi.python.org/pypi/piphttp://pypi.python.org/pypi/piphttp://pypi.python.org/pypi/setuptoolshttp://pypi.python.org/pypi/http://find/


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Installing NumPy, matplotlib and Biopython

Biopython relies on a few other Python packages for extrafunctionality. Well use these:

numpy efficient numerical functions and data structures(for Bio.PDB)

matplotlib plotting (for Bio.Phylo)

Then finally:

biopython the reason were here today

(Biopython, NumPy, matplotlib, setuptools and pip are also packaged for

many Linux distributions.)


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Testing

Check your Biopython installation:

>>> import Bio

>>> print Bio. version

Import a NumPy-based component:

>>> from Bio import PDB

Show a simple plot:

>>> from matplotlib import pyplot

>>> pyplot.plot(range(5), range(5))

>>> pyplot.show()


Sequences and alignments
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Lets start using


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Biopython1 Sequences and alignments

The Seq objectSeqIO and the SeqRecord object

2 NCBI EUtils and BLASTEUtils: Entrez Programming UtilitiesNCBI BlastExternal programs

3 Phylogenetics

4 Protein structures


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Sequencesand

Alignments


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The Seq object

>>> from Bio.Seq import Seq

>>> myseq = Seq(AGTACACTGGT)

>>> myseq

Seq(AGTACACTGGT, Alphabet())>>> print myseq

AGTACACTGGT

>>> myseq.transcribe()

Seq(AGUACACUGGU, RNAAlphabet())>>> myseq.translate()

Seq(STL, ExtendedIUPACProtein())


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q gNCBI EUtils and BLAST



A Seq object consists of:

data the underlying Python character string

alphabet DNA, RNA, protein, etc.

It supports most Python string methods:

>>> myseq.count(GT)2

And some biology-specific methods, too:>>> myseq.reverse complement()

Seq(ACCAGTGTACT, Alphabet())

Intrigued? Read on:>>> help(Seq)


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q gNCBI EUtils and BLAST



SeqIO: Sequence Input/Output

Sequence data is stored in many different file formats.Bio.SeqIO supports:

abi fastq phylip swissace genbank pir tab

clustal ig qual uniprot-xmlembl imgt seqxml

emboss nexus sff fasta phd stockholm

Manually fetch some data from the PDB website: 5

1ATP.fasta two protein sequences, FASTA format

1ATP.pdb the 3D structure, for later

5

http://www.rcsb.org/pdb/explore/explore.do?structureId=1ATPEric Talevich IOB Workshop: Biopython

http://www.rcsb.org/pdb/explore/explore.do?structureId=1ATPhttp://www.rcsb.org/pdb/explore/explore.do?structureId=1ATPhttp://find/


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NCBI EUtils and BLASTPhylogenetics

Protein structures


The SeqIO API

SeqIO provides four functions:

parse: Iteratively parse all elements in the file

read: Parse a one-element file and return the elementwrite: Write elements to a file

convert: Parse one format and immediately write another

Biopython uses the same I/O conventions for alignments(AlignIO), BLAST results (Blast), and phylogenetic trees(Phylo).


Sequences and alignmentsNCBI EU l d BLAST Th S b
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Protein structures


The SeqRecord object

SeqIO.parse returns SeqRecords.SeqRecord wraps a Seqobject and attaches metadata.

1 Pass the file name to the SeqIO parser; specify FASTA format:

from Bio import SeqIOseqrecs = SeqIO.parse("1ATP.fasta", "fasta")

print seqrecs


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Protein structures


The SeqRecord object

SeqIO.parse returns SeqRecords.SeqRecord wraps a Seqobject and attaches metadata.

1 Pass the file name to the SeqIO parser; specify FASTA format:

from Bio import SeqIOseqrecs = SeqIO.parse("1ATP.fasta", "fasta")

print seqrecs

2 To see all records at once, convert the iterator to a list:

allrecs = list(seqrecs)print allrecs[0]

print allrecs[0].seq


Sequences and alignmentsNCBI EUtils a d BLAST The Se object
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Protein structures


Example: Shuffled sequences

Given a real DNA sequence, create a background set ofrandomized sequences with the same composition.

Procedure:

1 Read the source sequence from a file Use Bio.SeqIO


Sequences and alignmentsNCBI EUtils and BLAST The Seq object


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Protein structures




Procedure:


2 In a loop:

Shuffle the sequence

Use random.shuffle from Pythons standard libraryCreate a new SeqRecord from the shuffled sequence

Because SeqIO.write works with SeqRecords




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Protein structures




Procedure:


2 In a loop:

Shuffle the sequence

Use random.shuffle from Pythons standard libraryCreate a new SeqRecord from the shuffled sequence

Because SeqIO.write works with SeqRecords

3 Write the shuffled SeqRecords to another file


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Protein structures


import randomfrom Bi o import SeqIO

from Bi o . Seq import Seqfrom Bio . SeqRe cord import SeqRecord

o r i g r e c = SeqIO . r e a d ( "gi2.gb", "genbank" )a l p h a b e t = o r i g r e c . s eq . a l p h a b e to u t r e c s = [ ]f o r i i n x r a n g e ( 1 , 3 1 ) :

n u c l e o t i d e s = l i s t ( o r i g r e c . s eq )random . s h u f f l e ( n u c l e o t i d e s )n e w s e q = S e q ( "" . j o i n ( n u c l e o t i d e s ) , a l p h a b e t )n e w r e c = S e q Re c o rd ( n e w s e q ,

i d="shuffle" + s t r ( i ) )o u t r e c s . a pp en d ( n e w r e c )

S eq IO . w r i t e ( o u t r e c s , "gi2_shuffled.fasta", "fasta" )


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C Ut s a d SPhylogenetics

Protein structures

e Seq objectSeqIO and the SeqRecord object

Example: ORF translation

Split a set of unannotated DNA sequences into uniqueORFs, translating in all 6 frames.

Biopython can help with each piece of this problem:

1 Parse the given unannotated DNA sequences (SeqIO.parse)2 Get the template strands sequence (Seq.reverse complement)

3 Translate both strands into protein sequences (Seq.translate)

4 Shift each strand by +1 and +2 for alternate reading frames

(string-like Seq slicing)5 Split sequences at stop codons (Seq.split(*))

6 Write translated sequences to a new file (SeqIO.write)


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q jSeqIO and the SeqRecord object

d e f t r a n s l a t e s i x f r a m e s ( s eq , t a b l e =1):

T r a n s l a t e a n u c l e o t i d e s eq ue nc e i n 6 f ra me s .

R e t u r n s an i t e r a b l e o f 6 t r a n s l a t e d p r o t e i ns e q u e n c e s .r e v = s e q . r e v e r s e c o m p l e m e n t ( )f o r i i n r a n ge ( 3 ) :

# C od in g ( C r i c k ) s t r a n d y i e l d s eq [ i : ] . t r a n s l a t e ( t a b l e )

# Te mp la te ( W atson ) s t r a n d y i e l d r e v [ i : ] . t r a n s l a t e ( t a b l e )


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SeqIO and the SeqRecord object

d e f t r a n s l a t e o r f s ( s eq ue nc es , m i n p r o t l e n =60):

F i nd and t r a n s l a t e a l l ORFs i n s e qu e nc e s .

T r a n s l a t e s e a c h s eq ue nc e i n a l l 6 r e a d i n g f r a m e s ,s p l i t s s e q u e n c es on s t o p c odo ns , and p r o d uc e s ani t e r a b l e o f a l l p r o t e i n s e q u e n c e s o f l e n g t h a t l e a s t m i n p r o t l e n .

f o r s e q i n s e q u e n c e s :

f o r f r a m e i n t r a n s l a t e s i x f r a m e s ( s e q ) :f o r p r o t i n f r a me . s p l i t ( "*") :

i f l e n ( pr ot ) >= m i n p r o t l e n :

y i e l d p r ot




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from Bi o import SeqIO

from Bio . SeqRe cord import SeqRecord

i f n a m e == "__main__" :import s y si n f i l e = s y s . s t d i no u t f i l e = s y s . s t d o u t

r e c o r d s = SeqIO . pa r se ( i n f i l e , "fasta" )s e q s = ( r e c . s e q f o r r e c i n r e c o r d s )p r o t e i n s = t r a n s l a t e o r f s ( s eq s )s e q r e c s = ( S eq Re co rd ( se q , i d="orf"+ s t r ( i ) )

f o r i , s e q i n e n u m e r a t e ( o r f s ) )

SeqIO . w r i t e ( s r e c s , o u t f i l e , "fasta" )



Ph l iThe Seq objectS IO d h S R d bj


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AlignIO and the Alignment object

Alignment: a set of sequences with the same length and alphabet.

Use AlignIO just like SeqIO:>>> from Bio import AlignIO

>>> aln = AlignIO.read("PF01601.sto", "stockholm")>>> print aln

SingleLetterAlphabet() alignment with 22 rows and 730 columnsNCTDAV-----LTYSSFGVCADGSIIA-VQPRNV-----SYDSV...HIQ Q1HVL3 CVH22/539-1170NCTTAV-----MTYSNFGICADGSLIP-VRPRNS-----SDNGI...HVQ SPIKE CVHNL/723-1356NCTEPV-----LVYSNIGVCKSGSIGY-VPSQS------GQVKI...HVQ Q692M1 9CORO/740-1383NCTEPA-----LVYSNIGVCKNGAIGL-VGIRN------TQPKI...HIQ Q0Q4F4 9CORO/729-1360NCTSPR-----LVYSNIGVCTSGAIGL-LSPKX------AQPQI...HVQ Q0Q4F6 9CORO/743-1371

NCTNPV-----LTYSSYGVCPDGSITR-LGLTD------VQPHF...--T A4ULL0 9CORO/726-1328NCTKPV-----LSYGPISVCSDGAIAG-TSTLQN-----TRPSI...KEW A6N263 9CORO/406-1035ECDIPIGAGICASYHTVSLLRSTSQKSIVAYTMS------LGAD...HYT Q6T7X8 CVHSA/647-1255...

DCE-PV-----ITYSNIGVCKNGAFVF-INVTH------SDGDV...HVH Q0PKZ5 CVPPU/797-1449



Ph l tiThe Seq objectS IO d th S R d bj t
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Snack Time



Phylogenetics

EUtils: Entrez Programming UtilitiesNCBI Blast
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External programs

EUtils and BLAST



Phylogenetics

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External programs

EUtils: Entrez Programming Utilities

Access NCBIs online services:from Bio import Entrez

Entrez.email = "[email protected]"



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External programs




Request a GenBank record:handle = Entrez.efetch(db="protein", id="69316",

rettype="gb", retmode="text")

record = SeqIO.read(handle, "gb")



Phylogenetics

EUtils: Entrez Programming UtilitiesNCBI BlastE l
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External programs




Request a GenBank record:handle = Entrez.efetch(db="protein", id="69316",

rettype="gb", retmode="text")

record = SeqIO.read(handle, "gb")

Specify multiple IDs in one query:handle = Entrez.efetch(db="protein",

id="349839,349840",

rettype="fasta", retmode="text")

records = SeqIO.parse(handle, "fasta")



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EUtils: Entrez Programming UtilitiesNCBI BlastE t l
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y gProtein structures

External programs

Interlude: SeqRecord attributes

seq: the sequence (Seq) itselfid: primary ID for the sequence, e.g. accession number

(string)

name: common name/id for the sequence, like GenBank

LOCUS iddescription: human-readible description of the sequence

letter annotations: restricted dictionary of additional info aboutindividual letters in the sequence, e.g. quality scores

annotations: dictionary of additional unstructured info

features: list ofSeqFeature objects with more structuredinformation e.g. position of genes on a genome,domains on a protein sequence.

dbxrefs: list of database cross-references (strings)



Phylogenetics

EUtils: Entrez Programming UtilitiesNCBI BlastExternal programs


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y gProtein structures

External programs

from Bi o import E n t r e z , Se qI OE n t r e z . e m a i l = "[email protected]"

h a n d l e = E n t r e z . e f e t c h ( d b="nucleotide", i d="M95169",r e t t y p e ="gb", r e t mo d e="text" )

r e c o r d = S eq IO . r e a d ( h a n d l e , "genbank" )h a n d l e . c l o s e ( )p r i n t r e c o r d

p r i n t r e c o r d . f e a t u r e s [ 1 0 ]s l i c e d = r e co r d [ 2 0 0 0 0 : ] # L a s t 25% o f t h e genome p r i n t s l i c e d

from Bi o . Seq import Seq

from Bi o . Al p hab et import g e n e r i c p r o t e i nt r a n s l a t i o n s = [ f . q u a l i f i e r s [ "translation" ]

f o r f i n r e c o r d . f e a t u r e s [ 1 : ] ]p r o t e i n s = [ Seq ( t [ 0 ] , g e n e r i c p r o t e i n )

f o r t i n t r a n s l a t i o n s ]



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Protein structuresExternal programs

NCBI Blast

BLAST can be used either standalone or through NCBIs server.

Online: >>> from Bio.Blast import NCBIWWW>>> result handle = NCBIWWW.qblast(

blastp, nr, query string)

Standalone: Legacy (blastall):>>> from Bio.Blast.Applications import

BlastallCommandline

>>> help(BlastallCommandline)

New hotness (Blast+):>>> from Bio.Blast.Applications importNcbiblastpCommandline

>>> help(NcbiblastpCommandline)



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Parsing BLAST output

BLAST produces reports in plain-text and XML format.

Biopython requests XML by default.

>>> from Bio.Blast import NCBIWWW, NCBIXML

>>> result handle = NCBIWWW.qblast(blastp,

... nr, query string)

>>> blast record = NCBIXML.read(result handle)

>>> print blast record



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# S e ar c h f o r hom ologs o f a p r o t e i n s eq ue nc e

from Bi o import SeqIOfrom B i o . B l a s t import NBCIWWW, NCBIXML

# Read and r e f o r ma t t he q ue ry s eq ue nc e s e q r e c = Se qI O . r e a d ( gi2.gb, gb )q u e r y = s e q r e c . f o r ma t ( fasta )

# Su bm it an o n l i n e BLAST q u e r y # ( T hi s t a k e s some t im e t o r un )r e s u l t h a n d l e = NCBIWWW. q b l a s t ( blastx, nr, q u er y )






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Protein structuresp g

# 1 . Sa ve t h e BLAST r e s u l t s a s an XML f i l e

w i th open ( aprotinin_blast.xml , w ) a s s a v e f i l e :s a v e f i l e . w r i t e ( r e s u l t h a n d l e . r e a d ( ) )

r e s u l t h a n d l e . c l o s e ( )

# NB : The BLAST r e s u l t h a nd l e can o n l y be r e ad on ce # R el oa d i t from t he f i l e

w i th open ( aprotinin_blast.xml ) a s r e s u l t h a n d l e :b l a s t r e c o r d = NCBIXML . r e a d ( r e s u l t h a n d l e )


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Protein structures



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Protein structures

# 2 . D i s p l a y a h i s to gr a m o f BLAST h i t s c o r e s

d e f g e t s c o r e s ( a l i g n m e n t s ) :f o r a l n i n a l i g n m e n t s :

f o r hsp i n a l n . hsp s :y i e l d hsp . s c o r e

s c o r e s = l i s t ( g e t s c o r e s ( b l a s t r e c o r d . a l i g n m e n t s ) )

# Draw t h e h i s t o gr a mimport p y l a bp y l a b . h i s t ( s c o r e s , b i n s =20)p y l a b . t i t l e ( " S co re s of % d B LA ST hi ts " % l e n ( s c o r e s ) )

p y l a b . x l a b e l ( "BLAST score" )p y l a b . y l a b e l ( " # hi ts " )p y la b . show ( )

# Save a copy f o r l a t e r p y l a b . s a v e f i g ( aprotinin_scores.png )



Protein structures



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Protein structures

Figure: Histogram of BLAST scores generatedbypylab



Protein structures

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Protein structures

# 3 . E x t r a c t t h e s eq ue nc es o f h i g hs c o r i n g BLAST h i t s

from Bi o . Seq import Seqfrom Bio . SeqRe cord import SeqRecord

d e f g e t h s p s ( a l ig n me n ts , t h r e s h o l d ) :f o r a l n i n a l i g n m e n t s :

f o r hsp i n a l n . hsp s :

i f h s p . s c o r e >= t h r e s h o l d :y i e l d S eq R ec or d ( Seq ( h sp . s b j c t ) ,

i d =a l n . a c c e s s i o n )break

b e s t s e q s = g e t h s p s ( b l a s t r e c o r d . a l i gn me n t s , 3 2 1)S eq IO . w r i t e ( b e s t s e q s , aprotinin.fasta, fasta )



Protein structures



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u u

Calling other external programs

Biopython has wrappers for other command-line programs in:

Bio.Blast.Applications the Blast+ suite

Bio.Align.Applications Muscle, ClustalW, . . .Bio.Emboss.Applications needle, water, . . .

Lets re-align our BLAST results using Muscle, and format the

alignment for use with stand-alone Phylip.



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from Bi o import A l i g n I Ofrom B io . A l i g n . A p p l i c a t i o n s import MuscleCommandlinefrom S t r i n g I O import S t r i n g I O

# C o n st r u ct t he s h e l l command m u s cl e c m d = M u sc l eC o mm a nd l in e ( i n p u t="aprotinin.fasta" )

# E x e c u t e t h e command # Get o ut pu t ( t he a l i gn m e nt ) and any e r r o r m es sa ge s

m u sc l e o u t , m u s c l e e r r = m usc le c md ( )

# Read t he a l i g n m e nt ba ck i na l i g n = A l i g n I O . r e a d ( S t r i n g I O ( m u s c l e o u t ) , "fasta" )

# Format t he a l i gn m e n t f o r P h y l i p

A l i g n I O . w r i t e ( [ a l i g n ] , aprotinin.phy, phylip )



Protein structures
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Phylogenetics



Protein structures
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Phylogenetic tree I/O

Start with:>>> from Bio import Phylo

Input and output of trees is just like SeqIO:

read, parse single or multiple trees in Newick, Nexus andPhyloXML formats

write to any of the formats supported by read/parse

convert between two formats in one step

Use StringIO to load strings directly:>>> from cStringIO import StringIO

>>> handle = StringIO("((A,B),(C,(D,E)));")

>>> tree = Phylo.read(handle, "newick")



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Whats in a tree?

Make a tree with branch lengths:>>> tree = Phylo.read(StringIO("((A:1,B:1):2,

... (C:2,(D:1,E:1):1):1);"), "newick")

View the object structure of the entire tree:>>> print tree

Draw an ASCII-art (plain text) representation:>>> Phylo.draw ascii(tree)

. . . OK, lets do it properly now:>>> Phylo.draw(tree)



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Modify the tree

Check the tree object for its methods:>>> help(tree)

Try a few:>>> tree.get terminals()>>> clade = tree.common ancestor("A", "B")

>>> clade.color = "red"

>>> tree.root with outgroup("D", "E")

>>> tree.ladderize()>>> Phylo.draw(tree)



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External applications

Biopython wraps a number of external programs for phylogenetics.Were not going to use them now, but heres where to find them:

Bio.Phylo.PAML PAML wrappers & helpers

Bio.Phylo.Applications command-line wrapper for PhyML(PhymlCommandline); RAxML and others on theway. (Anything youd like to see sooner?)

Bio.Emboss.Applications other tools ported via Embassy,

including Phylip



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Proteinstructures



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Going 3D: The PDB module

Load a structure:>>> from Bio import PDB

>>> parser = PDB.PDBParser()

>>> struct = parser.get structure(1ATP,

1ATP.pdb)



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Going 3D: The PDB module

Load a structure:>>> from Bio import PDB


>>> struct = parser.get structure(1ATP,

1ATP.pdb)

Inspect the object hierarchy:

>>> list(struct)

>>> model = struct[0]

>>> list(model)>>> chain = model[E]

>>> list(chain)

>>> residue = chain[15]

>>> list(residue)



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Figure: The SMCRA object hierarchy



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Extracting a peptide sequence

Get the amino acid sequence through a Polypeptide object:

>>> from Bio import PDB


>>> struct = parser.get structure(1ATP,... 1ATP.pdb)

>>> ppb = PDB.PPBuilder()

>>> peptides = ppb.build peptides(struct)

>>> for pep in peptides:

... print pep.get sequence()




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Calculating RMSD

Given two aligned structures, filter a list of targetresidues for high RMS deviation.

Input: list of residue positions (integers)two equivalent chains from aligned protein

models residue numbers must matchMinimum RMSD value (float)

Output: list of residue positions, filtered

Procedure: 1 Extract coordinates ofC atoms2

If available (not glycine), extractCcoordinates, too

3 Use Bio.SVDSuperimposer to calculate theRMSD between coordinates

4 Compare to the given RMSD threshold



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from Bio . SVDSup erimp oser import SVDSuperimposerfrom numpy import a r r a y

d e f f i l t r m s ( r e s i d s , r e f c h a i n , cmpchain , t h r e s h = 0. 5 ):s u p e r = S V DS u pe r i mp o se r ( )f o r r e s i n r e s i d s :

r e f r e s = r e f c h a i n [ r e s ]c m p r e s = c m p ch a i n [ r e s ]

c oo rd 1 = [ r e f r e s [ CA] . g e t c o o r d ( ) ]c o o r d 2 = [ c m pr e s [ CA] . g e t c o o r d ( ) ]i f r e f r e s . h a s i d ( CB ) and c m p r e s . h a s i d ( CB ) :

# Not g l y c i n e c o o r d 1 . a pp en d ( r e f r e s [ CB] . g e t c o o r d ( ) )

coo rd2 . append ( cmp res [ CB] . g e t c o o r d ( ) )s u p e r . s e t ( a r r a y ( c o o r d 1 ) , a r r a y ( c o o r d 2 ) )rmsd = s u p er . g e t i n i t r m s ( )i f rmsd >= t h r e s h o l d :

y i e l d r e s



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Figure: Superimposed structures, with selected deviating residues




F th di
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Further reading

Biopython tutorial:http:

//biopython.org/DIST/docs/tutorial/Tutorial.html

Biopython wiki:http://biopython.org/

This presentation:http://www.slideshare.net/etalevich/

biopython-programming-workshop-at-uga



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